Algal and Bacterial Nitrogen Processing in a Zero- Discharge Suspended-Culture Shrimp Production System

Transcription

1 Clemson University TigerPrints All Theses Theses Algal and Bacterial Nitrogen Processing in a Zero- Discharge Suspended-Culture Shrimp Production System Christian-dominik Henrich Clemson University, henrich@clemson.edu Follow this and additional works at: Part of the Aquaculture and Fisheries Commons Recommended Citation Henrich, Christian-dominik, "Algal and Bacterial Nitrogen Processing in a Zero-Discharge Suspended-Culture Shrimp Production System" (2008). All Theses This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact kokeefe@clemson.edu.

2 ALGAL AND BACTERIAL NITROGEN PROCESSING IN A ZERO DISCHARGE SUSPENDED CULTURE SHRIMP PRODUCTION SYSTEM A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Master of Science Biosystems Engineering By Christian Dominik Henrich May 2008 Title Page Accepted by: Dr. David Brune, Committee Chair Dr. Thomas Schwedler Mr. Kendall Kirk

3 ABSTRACT The objective of this research was to further modify and develop the Clemson Partitioned Aquaculture System (PAS) design resulting in a system design capable of yielding economic feasible production rates of marine shrimp, Litopenaeus vannamei, in excess of 45,000 (40,000 ) within a 5 month culture period while minimizing impact on the surrounding environment. The operation of the pilot scale high rate prototype shrimp culture system showed that it is possible to combine chemoautotrophic and photoautotrophic processing of feed and waste products within the system. A total of 23,400 (20,800 ) were harvested at the end of the season (149 days from PL 8/9 shrimp to an average harvest weight of 11 feed rate throughout the season was of (1645 ² (649 ). The average ). With peak feed rates ). The denitrification unit had the capacity to reduce, at a nitrate concentration of 47. At the end of the season the nitrification reactor had the capacity to nitrify up to 13.5 ² at 1000 nitrifying biomass concentration. A design for an optimal combined photoautotrophic/ chemoautotrophic system for high rate, zerodischarge culture of marine shrimp was proposed. This system is projected to provide a capacity to process a maximum nitrogen input of 12 ² yielding a ii

4 maximum feed application rate 2,343 projected shrimp yield of 87,889 growing season. (2,109 ), yielding a maximum (79,087 ) within a 150 day iii

5 ACKNOWLEDGEMENTS I would like to express my appreciation to my advisor, Dr. David Brune, for his guidance in this research and for his part in making my graduate work rewarding and enjoyable. I would like to thank the members of my committee, Dr. Thomas Schwedler and Mr. Kendall Kirk, for their advice, insights and review of my thesis. I would also like to thank Mr. Scott Davis and his farm crew, and Vickie Byko for their great assistance. I want to thank my Family and my friends, especially Christiane, Bianca and Axel, for all their encouragement and support. Last but not least, thanks to my officemates, to bear my curses. THANK YOU iv

6 TABLE OF CONTENTS Page TITLE PAGE... I ABSTRACT... II ACKNOWLEDGEMENTS... IV LIST OF TABLES... IX LIST OF FIGURES... XIV 1 INTRODUCTION Sustainable Agriculture and Aquaculture Research Objectives BACKGROUND Environmental Impact of Aquaculture Aquaculture and Fisheries Reducing Aquaculture Impacts Zero Discharge Aquaculture; The Partitioned Aquaculture System (PAS) Biological Nitrogen Transformation The Nitrogen Cycle v

7 2.3.2 Stoichiometry of Biological Nitrogen Transformations Microbial Processing of Nitrogen Microbial Populations in Aquaculture Systems Biofloc Communities in Aquaculture Processes METHODS PAS Configuration and Components Artificial Environment PAS Configuration and Water Circulation Aeration Methods for Data Collection Field Measurements Field Water Quality Determination Laboratory Water Quality Determination Operational Procedures Shrimp Stocking and Harvest Shrimp Feeding PH Control Water Exchange Sequencing Batch Reactor (SBR) Control Data Analysis and Manipulation Interpolations Re carbonation Rate Day and 7 Day Moving Averages Unionized Ammonia Carbon Mass Balance Equations Nitrogen Mass Balance Equations Shrimp Biomass Estimation RESULTS AND DISCUSSION Obtained Seasonal Data Outline Water Quality Summary vi

8 4.1.3 System Configuration and Operational Adjustments Photoautotrophic Dominated PAS Performance Chemoautotrophic Dominated PAS Performance Nitrification Reactor Performance Settling Tank/Denitrification Reactor Performance Settling Tank/Denitrification Reactor Sludge Analysis PROJECTED NITROGEN BALANCE AND PROPOSED SYSTEM DESIGN Projected Nitrogen Balance Proposed System Configuration SUMMARY AND CONCLUSIONS Summary Conclusions Shrimp Culture System Prototype Operation and Performance Proposed Shrimp System Configuration, Capacity, Yield, and Operation APPENDICES A. Abbreviations B. Symbols C. Unit Conversions D to 2005 PAS Configuration and Water Circulation vii

9 E. Obtained Data Shrimp Unit # F. Obtained Data Shrimp Unit # G. Obtained Data Tilapia confined Unit # H. Obtained Data Denitrification Reactor/Settling Tank (Unit #3A)151 I. Obtained Data Shrimp Unit # J. Obtained Data Nitrification Reactor LITERATURE CITED viii

10 LIST OF TABLES Table Page 1 Advantages and disadvantages of nitrogen processing by algae and nitrifyers Oxidation states of nitrogen species Major groups of microorganisms related to nitrogen transformation Critical water quality criteria for food application Critical water quality criteria for ph control SBR cycle settings (times) Characterization of 2007 PAS component system performance Average water quality and system parameters; Unit #1; day 43 through day 96 (Photoautotrophic dominated) Shrimp biomass and individual weights; Unit #1 day 43 through day 96 (Photoautotrophic dominated) Water flows and detention times; Unit #1, day 43 through day 96 (Photoautotrophic dominated) Average water quality and system parameters; Unit #2; day 43 through day 96 (Photoautotrophic dominated) Shrimp biomass and individual weights; Unit #2 day 43 through day 96 (Photoautotrophic dominated) ix

11 Table Page 13 Water flows and detention times; Unit #2, day 43 through day 96 (Photoautotrophic dominated) Average water quality and system parameters; Unit #4; day 43 through day 96 (Photoautotrophic dominated) Shrimp biomass and individual weights; Unit #4 day 43 through day 96 (Photoautotrophic dominated) Water flows and detention times; Unit #4, day 43 through day 96 (Photoautotrophic dominated) Carbon mass balance summary; Unit #1; day 43 through day 96 (Photoautotrophic dominated) Nitrogen mass balance summary; Unit #1; day 43 through day 96 (Photoautotrophic dominated) Carbon mass balance summary; Unit #2; day 43 through day 96 (Photoautotrophic dominated) Nitrogen mass balance summary; Unit #2; day 43 through day 96 (Photoautotrophic dominated) Carbon mass balance summary; Unit #4; day 43 through day 96 (Photoautotrophic dominated) Nitrogen mass balance summary; Unit #4; day 43 through day 96 (Photoautotrophic dominated) x

12 Table Page 23 Average water quality and system parameters; Unit #1; day 116 through day 137 (Bacterial dominated) Shrimp biomass and individual weights; Unit #1 day 116 through day 137 (Bacterial dominated) Water flows and detention times; Unit #1, day 116 through day 137 (Bacterial dominated) Average water quality and system parameters; Unit #2; day 126 through day 137 (Bacterial dominated) Shrimp biomass and individual weights; Unit #2 day 126 through day 137 (Bacterial dominated) Water flows and detention times; Unit #2, day 126 through day 137 (Bacterial dominated) Average water quality and system parameters; Unit #4; day 116 through day 137 (Bacterial dominated) Shrimp biomass and individual weights; Unit #4 day 116 through day 137 (Bacterial dominated) Water flows and detention times; Unit #4, day 116 through day 137 (Bacterial dominated) Carbon mass balance summary; Unit #1; day 116 through day 137 (Bacterial dominated) xi

13 Table Page 33 Nitrogen mass balance summary; Unit #1; day 116 through day 137 (Bacterial dominated) Carbon mass balance summary; Unit #2; day 126 through day 137 (Bacterial dominated) Nitrogen mass balance summary; Unit #2; day 126 through day 137 (Bacterial dominated) Carbon mass balance summary; Unit #4; day 116 through day 137 (Bacterial dominated) Carbon mass balance summary; Unit #4; day 116 through day 137 (Bacterial dominated) TAN and nitrite balance for the nitrification reactor (days 43 96) TAN and nitrite balance for nitrification reactor (days ) Independently determined nitrification reactor oxidation rates (days 137, 147 and 156) Denitrification reactor carbonate mass balance (days ) Denitrification reactor nitrate mass balance (days 129 day 137) Proposed 1 ha shrimp production system component areas and volumes Past and proposed shrimp yield, season length and feed rates Abbreviations Symbols xii

14 Table Page 47 Indices Metric to American unit conversions xiii

15 LIST OF FIGURES Figure Page 1 General principle of an aquaculture pond without supplemental wastewater treatment, with (2) and without (1) discharge General principle of an aquaculture pond with external wastewater treatment Benefits of biological wastewater treatment Aquaculture pond system with enhanced internal biological wastewater treatment General nitrogen cycle in nature Microbial populations and water quality interactions Microscopic view of brown biofloc with blue green algae (left) compared to green algae (right) The PAS greenhouse enclosure PAS greenhouse enclosure components PAS nitrification reactor Individual PAS unit for shrimp culture Early 2007 PAS configuration Final 2007 PAS configuration Aeration distribution of 2007 Clemson PAS Water exchange control circuit diagram xiv

16 Figure Page 16 SBR control circuit diagram Shrimp feed intake in % of average body weight dependent of their average body weight at optimum growth conditions Carbon mass balance for the photoautotrophic dominated units, day 43 through day Nitrogen mass balance for the photoautotrophic dominated units, day 43 through day Carbon mass balance; Chemoautotrophic dominated, units #1 and #4, day 116 through day Nitrogen mass balance; Chemoautotrophic dominated, units #1 and #4, day 116 through day Carbon mass balance; Chemoautotrophic dominated; Photoautotrophic assisted, unit #2, day 116 through day Carbon mass balance; Chemoautotrophic dominated; Photoautotrophic assisted, unit #2, day 116 through day Alkalinity mass balance; Chemoautotrophic dominated, unit #1, day 116 through day Alkalinity mass balance; Chemoautotrophic dominated, unit #4, day 116 through day Average nitrate mass balance for unit #3; Mass balance day 129 through day 137 (Denitrifying) xv

17 Figure Page 27 TKN versus depth in Settling Tank/Denitrification reactor Solids concentration versus depth in Settling Tank/Denitrification reactor Flow path and sludge elevation in Settling Tank/Denitrification Projected average nitrogen balance for optimal combined photoautotrophic/ chemoautotrophic PAS culture of marine shrimp Proposed optimal PAS photoautotrophic/ chemoautotrophic system design PAS configuration PAS configuration PAS configuration Event timeline for unit # Food input rate for unit # Water circulation chart from unit # Water exchanges per day unit # Daily and 7 day average temperature in unit # Daily and 7 day average dissolved oxygen concentration in unit # Daily and 7 day average salinity in unit # Daily and 7 day average ph in unit # Daily and 7 day average secchi depth in unit # xvi

18 Figure Page 44 Weekly Solids in unit # Alkalinity and NaHCO 3 supplement in unit # Daily and 7 day average total ammonia nitrogen in unit # Daily and 7 day average unionized ammonia in unit # Daily and 7 day average nitrite in unit # Daily and 7 day average nitrate in unit # Total Kjeldahl and particulate organic nitrogen in unit # Particulate carbon nitrogen ratios(c:n ratio) in unit # Net algal photosynthesis and water column respiration in unit # Shrimp density projected from food consumption and measured shrimp size in unit # Projected shrimp biomass in unit # Event timeline for unit # Food input rate for unit # Water circulation chart from unit # Water exchanges per day in unit # Daily and 7 day average temperature in unit # Daily and 7 day average dissolved oxygen concentration in unit # Daily and 7 day average salinity in unit # Daily and 7 day average ph in unit # Daily and 7 day average secchi depth in unit # xvii

19 Figure Page 64 Weekly Solids in unit # Alkalinity and NaHCO 3 supplement in unit # Daily and 7 day average total ammonia nitrogen in unit # Daily and 7 day average unionized ammonia in unit # Daily and 7 day average nitrite in unit # Daily and 7 day average nitrate in unit # Total Kjeldahl and particulate organic nitrogen unit # Particulate carbon nitrogen ratios (C:N ratio) in unit # Net algal photosynthesis and water column respiration in unit # Shrimp density projected from food consumption and measured shrimp size in unit # Projected shrimp biomass in unit # Event timeline for unit # Water circulation chart from unit # Water exchanges per day in unit # Daily and 7 day average temperature in unit # Daily and 7 day average dissolved oxygen concentration in unit # Daily and 7 day average salinity in unit # Daily and 7 day average ph in unit # Daily and 7 day average secchi depth in unit # Weekly Solids in unit # xviii

20 Figure Page 84 Alkalinity and NaHCO 3 supplement in unit # Daily and 7 day average total ammonia nitrogen in unit # Daily and 7 day average unionized ammonia in unit # Daily and 7 day average nitrite in unit # Total Kjeldahl and particulate organic nitrogen unit # Particulate carbon nitrogen ratios in unit # Net algal photosynthesis and water column respiration in unit # Event timeline for unit #3A Daily and 7 day average total ammonia nitrogen in unit #3A Daily and 7 day average nitrite in unit #3A Event timeline for unit # Food input rate for unit # Water circulation chart from unit # Water exchanges per day in unit # Daily and 7 day average temperature in unit # Daily and 7 day average dissolved oxygen concentration in unit # Daily and 7 day average salinity in unit # Daily and 7 day average ph in unit # Daily and 7 day average secchi depth in unit # Weekly Solids in unit # Alkalinity and NaHCO 3 supplement in unit # xix

21 Figure Page 105 Daily and 7 day average total ammonia nitrogen in unit # Daily and 7 day average unionized ammonia in unit # Daily and 7 day average nitrite in unit # Daily and 7 day average nitrate in unit # Total Kjeldahl and particulate organic nitrogen unit # Particulate carbon nitrogen ratios (C:N ratio) of unit # Net algal photosynthesis and water column respiration in unit # Shrimp density projected from food consumption and measured shrimp size in unit # Projected shrimp biomass in unit # Event timeline for the reactor Water circulation chart from the reactor Water exchanges per day in the reactor Daily and 7 day average temperature in the reactor Daily and 7 day average dissolved oxygen concentration in the reactor Daily and 7 day average salinity in the reactor Daily and 7 day average ph in the reactor Weekly Solids in the reactor Daily and 7 day average total ammonia nitrogen in the reactor Daily and 7 day average total ammonia nitrogen in the reactor discharge xx

22 Figure Page 124 Daily and 7 day average unionized ammonia in the reactor Daily and 7 day average nitrite in the reactor Daily and 7 day average nitrite in the reactor discharge xxi

23 Introduction 1 INTRODUCTION 1.1 Sustainable Agriculture and Aquaculture Sustainability is defined as: Development, meeting the needs of the present without compromising the ability of future generations to meet their needs. (WSDE, 2008) In the case of Agriculture, this implies feed and food production at renewable capacity, within the capacity of the producing environment, with assimilation, and recovery of waste byproducts. Sustainability may be measured by land, water and energy use, and protein transformation. To meet this objective, production must be tied to solar energy inputs, to the greatest extent possible, with maximum by product recovery and internal nutrient recycling. Aquaculture, one of the most rapidly developing areas of agriculture, offers the greatest potential for utilization of high rate of photosynthesis because of the rapid growth potential of microalgal populations within aquaculture systems. It is this potential that was targeted for development, optimization and application through continued improvement in the design of Clemson s recently patented intense aquaculture technology, the Partitioned Aquaculture System (PAS). Page 1

24 Introduction 1.2 Research Objectives The overall objective of this research was the further modification and development of the PAS design capable of yielding production of shrimp biomass in excess of 45,000 kg/ha (40,000 lb/acre) within a 5 month culture period while minimizing impact on the surrounding environment. Specific sub objectives include: a) To quantify the physical chemical and microbial processing rates of feed and waste products within the system, b) To develop a quantitative understanding of interactions of algal and bacterial communities within the system, c) To develop designs of a high rate PAS culture system capable of optimal utilization of bacterial and algal processing of waste products, maximizing system carrying capacity, while eliminating waste discharges. Specific research tasks undertaken to meet these objectives included: a) Operation of pilot scale high rate prototype shrimp culture system, b) Collection, reduction and analysis of data from system operation, c) Characterize system nitrogen mass balances and rates of transformation, d) Propose a design of an optimal combined photoautotrophic /chemoautotrophic system for high rate, zero discharge culture of marine shrimp. Page 2

25 Background 2 BACKGROUND 2.1 Environmental Impact of Aquaculture Aquaculture and Fisheries The demand for seafood increases with population and income growth. Conventional fisheries are already experiencing declining fishing stocks (Guillotreau, 2004) and are also highly destructive to marine environments, such as coral reefs and highly diversified sediments. The growing seafood demand is increasing the need for aquaculture solutions to satisfy this need (Josupeit, 2004). However, intensive aquaculture systems can cause a devastating impact on the environment. Typically, only a portion of the applied feed is assimilated by the targeted species, the rest is excreted into the water column. Flow through systems, with high water exchange and production capacity also discharge large quantities of polluted wastewater to the environment (US Environmental Protection Agency, 2000; Sharpley et al., 1999). Such discharges and impacts have already led to instances of collapse of poorly designed intensive shrimp aquaculture industries (Liao (1992), and Wang et al.(1995)). Untreated wastewater discharge (high total nitrogen, high BOD, high phosphorous, etc.) leads to eutrophication and low dissolved oxygen levels. Algal blooms and followed by bloom decay can result in massive fish kills with collapse of the aquatic ecosystem. Ultimately Page 3

26 Background the pollutants impact humans and other animals that depend on clean water (US Environmental Protection Agency, 2000). In an attempt to improve aquaculture sustainability recent research has targeted the development of high intensity growout systems with no water discharge. Numerous research and development efforts have been directed at improving environmental sustainability, economics, and yield of limited or zero discharge aquaculture systems (Hopkins et al., 1993; Green et al., 1995; Sandifer et al., 1996; Brune et al., 2003, 2004; Burford, 2003; Wang, 2003; Schneider et al., 2007) Reducing Aquaculture Impacts Reduced Carrying Capacity and Water Discharge Within low yield aquaculture systems animal density is maintained at levels within the assimilative capacity of the system. Since no additional wastewater treatment is provided, the sediment and water column must provide the capacity to maintain water quality at sub lethal levels. In such cases marketable animal yields are low. Increasing production requires the addition of water exchange water with the external environment to maintain water quality at acceptable levels. This option, of course, transfers the pollution to pubic waters resulting in environmental impacts previously discussed in chapter This approach is illustrated in Figure 1. Page 4

27 Background (1)Pond No water exchange Nutrient recycle Extensive River (2)Pond High water exchange High environmental impact No nutrient recycle Semi intensive to intensive Estuary Figure 1: General principle of an aquaculture pond without supplemental wastewater treatment, with (2) and without (1) discharge. These extensive, semi extensive approaches are usually limited to fish and shrimp yields of 500 (extensive), to 5,000 (semi intensive) as opposed to 20,000 (intensive) shrimp yields (Shrimp News International, 2008). Physical Chemical Waste Treatment in Aquaculture Ozone foam fractionation, drum filtering, Ion exchange and other more energy intensive techniques are available to clean water. (Crittenden, 2005). This approach to aquatic animal culture is both capital and operational cost intensive. However, intensive recirculating aquaculture systems may be used to support culture of higher value animals. This approach is illustrated in Figure 2. Page 5

28 Background Waste water treatment system Producing sludge wastes High energy needs High area requirements High labor requirements Pond High water exchange Reduce environmental impact Intensive to super intensive Figure 2: General principle of an aquaculture pond with external wastewater treatment. These systems can be suitable and cost effective, with higher value animals requiring a higher degree of water quality control. This production design generally requires applications involving freshwater animals where saltwater corrosion is not an issue (Timmons 2007). This solution would be feasible for intensive farming, up to 20,000 or super intensive farming with up to 100,000 yield (Shrimp News International, 2008). Biological Waste Treatment in Aquaculture Biological treatment of municipal and industrial wastewaters has been well defined (Oswald, 1990; Reynolds, 1995; Crittenden, 2005). These techniques can be employed to intensify aquaculture production reducing environmental impacts, and potentially Page 6

29 Background improving long term sustainability. The key is to replace natural environment functions, thereby replacing natural processing of key limiting pollutants (like nitrogen), with more intensive treatment processes. Benefits provided with such systems are illustrated in Figure 3. Controlled wastewater treatment Nitrogen removal Beneficial additional organics removal Microorganisms use pollutants for growth Use of the natural ly occurring eutrophication process Microbial metabolism to remove wastes from aquacultural wastewater Beneficial additional solids removal Beneficial additional recycling of minerals Figure 3: Benefits of biological wastewater treatment. To employ managed microbial populations to process pollutants from aquaculture systems, it is necessary to understand the microorganisms involved, their impact on water quality, and their natural habitat. Ammonia is one of the critical limiting metabolites resulting from aquatic processing of feed inputs, through fish metabolism, and excretion (Hargreaves, 1998). Proper engineering solutions focus on designed nitrogen cycles within the aquaculture system, to maximize processing of nitrogen Page 7

30 Background compounds, resulting in highly productive and hopefully, cost effective fish production. This approach is illustrated in Figure 4. Pond system with WW Treatment Internal water processing Reduced environmental impact Nutrients recycle Internal WW treatment Figure 4: Aquaculture pond system with enhanced internal biological wastewater treatment. This principle is employed in the Clemson PAS, which is discussed in this work. 2.2 Zero Discharge Aquaculture; The Partitioned Aquaculture System (PAS) Clemson s Partitioned Aquaculture System (US Patent ) was originally developed for production of channel catfish (Ictalurus punktatus). The PAS designs were shown to be capable of fivefold increased catfish yield as compared to conventional catfish production, and cost of production reductions of 5 to 10 cents per pound (Brune, 2003). The concept of the PAS is to divide the pond into several compartments with distinct physical, biological, and chemical processes. In the case of the PAS used for catfish production, the pond was reconfigured to contain a fish raceway with the Catfish (comprising 5% of total pond area), a raceway containing filter feeder organisms that Page 8

31 Background consume algae, and shallow basins for wastewater treatment used to grow algae (95% of total pond area). The key was to confine the Catfish at high densities while maintaining water circulation through the three compartments with low speed paddlewheels and supplemental oxygenation provided with fountain aerators located in the fish raceways only. The concept of growing shrimp in a PAS is similar, except shrimp must be distributed throughout the pond, and therefore, requires total pond oxygenation. A prototype system for shrimp growth trials was constructed at Clemson University Aquaculture facility in 2002 and growth trials were performed in 2003 (Brune 2004), 2004 (Kirk 2004), 2005 and 2007 (this work). The PAS was used to maintain habitable water quality conditions with zero water discharge during these years. As the PAS configuration was modified over the years, shrimp yields rose from 16,500[kg/ha] in 2003 (80% survival), 25,600[kg/ha] in 2004 (61% survival), to 37,400[kg/ha] in 2005 (51% survival). In 2003 an average of 180[kg feed/ha/day] was applied to the system, 424 [kg feed/ha/day] in 2004 and 674[kg feed/ha/day] in At the low feed rates in 2003, the waste removal system was algal dominated. In 2004 a nitrification/denitrification compartment in the back of the units was installed and solids were removed to improve nitrogen handling in the system, the units remained algal dominated (photosynthetic) for the first half of the season. In 2005, due to further increases in feed application rates and therefore higher nitrogen inputs to the system, the shrimp production units became bacterial dominated (chemoautotrophic/ heterotrophic). Based on the experiences gathered through the years of research, the Page 9

32 Background goal in 2007 was to combine the benefits of increased algal productivity within the shrimp units with high nitrogen processing through nitrification in a separate reactor. Advantages and disadvantages of both systems are shown in Table 1. Table 1: Advantages and disadvantages of nitrogen processing by algae and nitrifyers. ALGAE supplemented GREEN system BIOFLOC driven BROWN system positive negative positive Negative Photosynthesis (O 2 Production) Improved Shrimp health CO 2 injection Limited by light Limited uptake capacity High biomass yield Solids removal (or N recycling) Populations can be unstable Nitrification Ammonification of organic N Uptake of organic carbon Low biomass yield Readily settable solids Oxygen demand (high aeration costs) Carbonate supplement needed (because of alkalinity destruction) 2007 Solution: mixed system by concentrating Bioflocs in SBR Page 10

33 Background 2.3 Biological Nitrogen Transformation The Nitrogen Cycle The fate of nitrogen in nature is of particular interest and is presented in Figure 5, The nitrogen cycle. Nitrogen exists in several oxidation states and these oxidation states in the environment are controlled almost exclusively by microbial populations. The oxidation states of nitrogen species are shown in Table 2. Table 2: Oxidation states of nitrogen species. Oxidation state Nitrogen species Formula III Ammonium/ organic N NH + 4 / R NH 3 II I Hydroxylamine NH 2 OH ±0 Nitrogen gas N 2 +I Nitroxyl /Nitrous oxide NOH /N 2 O +II Nitric oxide NO +III Nitrite NO 2 +IV +V Nitrate NO 3 Source: Brock at al., Nitrogen gas is the energetically most stable species and is the major reservoir of nitrogen on earth. Some plants, Cyanobacteria, Rhizobium and Acetobacter in the soil and root nodules of legumes, fix nitrogen gas and transforming it into organic nitrogen compounds. Decomposition of organic matter by bacteria and fungi and excretion by animals releases organic N as ammonia nitrogen and urea. Plants, algae and bacteria can assimilate ammonia and use it as nitrogen source for biomass yield. Other bacteria like Nitrosomonas species and Nitrobacter species transform ammonia into nitrite and Page 11

34 Background nitrite into nitrate respectively using the reactions as a source of energy. This process of nitrification occurs under aerobic conditions. Nitrate is assimilated by plants or algae, or converted back into nitrogen gas by denitrification through several reaction steps occurring with an anoxic environment (BROCK et al., 1997). Some microorganisms are capable of oxidizing ammonia anaerobically (Anammox) to nitrogen gas at high ammonia concentrations (STROUS et al., 1999). Figure 5 illustrates the general nitrogen cycle, primary nitrogen species, and nitrogen metabolism pathways and microorganisms. Nitrogen fixers (Bacteria in root nodules, and soil) Gaseous Nitrogen (N 2 ) Denitrifying Bacteria (Anaerobic) Organic Nitrogen (R Nx) Nitrogen assimilators (Plants, algae, bacteria) Nitrate (NO 3 ) Nitrifying Bacteria (Aerobic) Nitrite (NO 2 ) Decomposers (Aerobic and anaerobic bacteria and fungi Ammonium Ion (NH 4 + ) and Ammonium (NH 3 ) Nitrifying Bacteria (Aerobic) Figure 5: General nitrogen cycle in nature. Source: Redrawn from BROCK et al., Page 12

35 Background Stoichiometry of Biological Nitrogen Transformations Nitrogen Fixation Nitrogen fixation in the root nodules of legumes is carried out by symbiotic bacteria (for example Clostridium posteurianum), and several anoxygenic phototrophic bacteria (Brock et al., 1997). + N 8 NH + H (1) 2 + H + 8e These microbes and process are not relevant to aquatic system applications. Heterotrophic Assimilation and Mineralization Nitrogen assimilation is used as nitrogen source by terrestrial plants, algae and bacteria, where ammonia and/or nitrate serves for growth. These nitrogen sources are converted into organically bound nitrogen in proteins and other organic molecules (Brock et al., 1997). NH NO 3 3 energy organic N energy organic N (Assimilation) (2) organic N NH 3 (Ammonification) When organisms decay and their biomass decomposes by anaerobic and aerobic bacteria and fungi organic molecules will be deaminated resulting in ammonia releases. Page 13

36 Background Nitrification Nitrosomonas and Nitrobacter species together oxidize ammonia to nitrite and then to nitrate yielding energy supporting the microbial metabolism (Brock et al., 1997). NH 3 + O + 2H NH OH + H O e NO 2 NH OH + H O + 5H e 2 (Ammonia oxidation) (3) NO + H O NO + 2e + 2H (Nitrite oxidation) NH + O + H O NO + 4e + 5H (Overall nitrification process) Nitrification consumes oxygen and therefore occurs only in the presence of aerobic conditions. Denitrification In the denitrification process, nitrate is reduced under anaerobic conditions to nitrogen gas through nitrite and other intermediates during anaerobic respiration, where nitrate is used as the electron acceptor (BROCK et al., 1997). NO NO + 2e + 2e 2NO + 2e N O + 2e H + 2H + 2H + 2H NO N O + H O N H NO + H + H O 2 O O (4) + 1 NO3 + 6 e + 7H N 2 + 3H 2O 2 (Overall denitrification process) Page 14

37 Background 2.4 Microbial Processing of Nitrogen Microbial Populations in Aquaculture Systems The major groups of microorganisms of interest for aquaculture, are photoautotrophs, chemoautotrophs, heterotrophs and denitrifiers. Table 3 illustrates the impacts these microorganisms produce on aqueous nitrogen species and their carbon source. Table 3: Major groups of microorganisms related to nitrogen transformation. Nitrogen uptake by photoautotrophic organisms (algae/ plants) Photosynthesis for energy Inorganic carbon source (CO 2 ) Assimilation of NH 4 or NO 2 (N source) Nitrification by chemoautotrophic bacteria (bacterial community) Oxidation of NH 4 and NO 2 (e donor) Inorganic carbon source Reduction of oxygen (e acceptor) Nitrogen assimilation by heterotrophic bacteria (bacterial community) Assimilation of NH 4 + (N source) Organic carbon (e donor) Ammonification of organic N (add NH 4 + ) Reduction of oxygen (e acceptor) Denitrification by heterotrophic denitrifiers (bacterial community) Source: Data according to Brock at al., Reduction of NO 3 = (e acceptor) Carbon source mainly organic (e donor) Page 15

38 Background No natural systems contain only one group of microorganisms, however one group of microorganisms may dominate the microbial ecology, depending on system conditions (Wyk et al. 2007) ) Biofloc Communit ties in Aquaculture Processess Bioflocs are clusters of insoluble solids, polymers, dead cells and inert provide surfacess for microorganisms to attach to and grow. Different matter, and studies have investigated the composition of bioflocs in aquaculture systems, as impacted by water column C:N ratio, nitrogen concentration and species, temperature, oxygen concentration and BOD level. A dynamic community of bacteria, diatoms, cyanobacteria, dinoflagellat es, zooplankton, and green algae are found in bioflocs (Leffler et al., 2007). A representation of a biofloc community is illustrated in Figure 6. Figure 6: Microbial populations and water quality interactions. Page 16

39 Background Figure 7 illustrates a typical biofloc structure observed in 2007 PAS operation. This sample contains mainly brown biofloc material together with blue green algae vs. a sample containing green algae as dominating organism. Figure 7: Microscopic view of brown biofloc with blue-green algae (left) compared to green algae (right). Page 17

40 Methods 3 METHODS 3.1 PAS Configuration and Components Artificial Environment A detailed description of the artificial environment construction was performed by Kirk (2004) and is summarized in this chapter. Fresh water was pumped into a concrete lined pond system to create the artificial marine environment. Red Sea brand evaporated seas salt was added once after the filling process. Despite of zero discharge operation and low permeability of the system, limited percolation required small additional supplement of salt during the growing season to maintain a target salinity of 12 [g/l]. The pond system was covered with a greenhouse, to protect shrimp from predatory birds and aquatic insects, and to control temperature during colder fall and spring months. But a semitransparent plastic covering of the greenhouse still allows light transmittance for photosynthesis. The greenhouse was completed in spring 2002 at the Calhoun Filed Research Laboratory on the Clemson University Campus, and is shown in Figure 8. Four concrete lined raceways with a total area of ¼ ac (0.1 ha) with each unit being 120 ft (36.6 m) in length and 22 ft (6.7 m) in width, with 3 ft (0.9 m) high side walls are covered under the greenhouse (see Figure 9). It is possible to open or close the greenhouse sides to adjust the temperature inside the greenhouse to maintain an optimal temperature of C, for shrimp growth and survival. Page 18

41 Methods Figure 8: The PAS greenhouse enclosure. Source: Brune, Figure 9: PAS greenhouse enclosure components. Source: Kirk, Page 19

42 Methods In 2007 a nitrification reactor was added in the back of the greenhouse consisting of a tank 40 ft (12.2 m) in length, 13 ft (3.9 m) wide and average depth of 10.5 ft (3.2 m). This reactor does not have an enclosure. A picture of the reactor is shown in Figure 10. Figure 10: PAS nitrification reactor. Source: Brune, Page 20

43 Methods PAS Configuration and Water Circulation To maintain homogeneous sediment mixing and water distribution, each unit is equipped with a six blade paddlewheel, 4 ft (1.2 m) diameter. The paddlewheels are driven by a variable speed electric motor with one motor driving two paddles. At the front of each unit an L shaped channel was constructed and the paddlewheel is placed at the end of the channel. A black plastic polyethylene curtain runs from the end of the channel through the middle of the unit, to route the flow in an ovular circuit (Kirk, 2004). The sketch of a single shrimp unit is presented in Figure 11. F l o w L shaped concrete wall Flow Plastic curtain In flow Paddlewheel Flow Outflow Figure 11: Individual PAS unit for shrimp culture. Source: Redrawn from to KIRK, The PAS configuration of the years is shown in appendix D. Page 21

44 Methods In 2007 a deep nitrification reactor was added to the back side of the greenhouse. The purpose was to investigate the potential for increasing algal fixation and nitrification and denitrification to increase the processing of nitrogen in the system. The anoxic settling tank/denitrification reactor (#3A/#3C) was increased in size and the tilapia containment zone was decreased in size. Figure 12: Early 2007 PAS configuration. Page 22

45 Methods At the end of the 2007 season the nitrification reactor was modified to be operated as sequencing batch reactor with 60 min fill, 20 min reaction, 20 min settle, and 20 min decant cycles rather than a two pond system with sludge recycling. Unit #2 was exchanging with the denitrification reactor/settling tank, the tilapia confined unit, and the nitrification reactor, units #1 and #4 were isolated. The final 2007 PAS configuration is shown in Figure 13. Figure 13: Final 2007 PAS configuration. Page 23

46 Methods Aeration Five ½ hp (373 W) regenerative blowers supplied 36 diffusers, equally distributed among the shrimp units. Additionally two 1.5 hp (1119 W) regenerative blowers supplied 22 diffusers, equally distributed among the shrimp units. During the growth season up to six 3/4 hp (559 W) Powerhouse aerators per unit where installed. Three Powerhouse aerators where installed in the tilapia confined zone (unit #3) and two in the nitrification reactor. When the nitrification reactor was changed to an SBR configuration, an additional 1.5 hp (1119 W) aerator was added to mix and aerate this reactor. Figure 14 shows the aeration distribution of the 2007 PAS system. Page 24

47 Methods Figure 14: Aeration distribution of 2007 Clemson PAS. Page 25

48 Methods 3.2 Methods for Data Collection Field Measurements Flow Rates The rate of water exchange between units was determined by capturing water in bucket calibrated to contain 16 kg of water. Time to fill was recorded. The flow rate in [gal/min] was then calculated using equation (5) ³ (5) With the density of water 1000, the calibrated weight of 16 in [kg] and the ³ measured time to fill to 16 kg line in [s]. Depth and Secchi depth The pond depth was measured with a stick 2 meters long, calibrated at 1 centimeter increments. The depth s where measured at the same location for every measurement. Photosynthesis One dark and one clear 300(ml) BOD bottle were filled with sample and placed on the water surface. The Oxygen was measured with an YSI BOD probe every 6 hours during a 24 hour period. Every time oxygen was measured, new sample water was filled into the bottles. The oxygen measurements from the dark bottle represent water column respiration, and from the light bottle represent net photosynthesis. Page 26

49 Methods Field Water Quality Determination Temperature Temperature measurements were obtained using YSI 550A DO/ Temperature monitor. The Temperature was measured at the same location for every measurement at approximately 30 cm below water surface. PH The ph measurements where obtained using a Pinpoint TM PH Monitor obtained from AQUATIC ECO SYSTEMS, INC. The ph was measured at the same location for every measurement at approximately 30 cm below water surface. DO The dissolved oxygen (DO) was measured using an YSI 550A DO/ Temperature monitor. The DO was measured at the same location for every measurement at approximately 30 cm below water surface. Salinity The salinity was measured with a Pinpoint TM Conductivity meter obtained from AQUATIC ECO SYSTEMS, INC. The conductivity was measured at the same location for every measurement at approximately 30 cm below water surface. Page 27

50 Methods Alkalinity The alkalinity was measured by titration of 0.2N H 2 SO 4 into 50(mL) of raw or diluted sample until ph of 4.5 was reached. The alkalinity concentration was calculated using equation (6). 20 (6) Nitrogen Field measurements of nitrogen concentrations were determined with HACH kits using the reagents and procedures summarized below: TAN (Total ammonia nitrogen) To measure ammonia nitrogen concentrations on site, an ammonia nitrogen Test Kit, HACH, Cat No (Model NI 8), with Nesslers reagent, Rochelle Salt, specified color viewing tubes, and color wheel to determine the concentrations was used. The range of this measurement kit was 0 to3 (mg N/L). 5(mL) of raw or diluted sample were filled into the test tubes and one drop of Rochelle salt was added and allowed to react for two minutes. Three drops of Nesslers reagent were mixed into to the sample and allowed to react for five minutes. A second test tube with 5(mL) sample without reagents (blank) was prepared. Then the two test tubes were used with the color wheel to determine the ammonia N (TAN) concentration. Page 28

51 Methods NO2 N (Nitrite) To measure nitrite nitrogen concentrations on site an nitrite nitrogen Test Kit, HACH Cat No (Model NI 15), with NitriVer 6 Nitrite Reagent Powder Pillows, specified color viewing tubes, and color wheel to determine the concentrations was used. The range of this measurement kit was 0.0 to 0.50 (mg N/L). 5(mL) of raw or diluted sample were filled into the test tubes and one NitriVer 6 Nitrite Reagent Powder Pillow was mixed in and allowed to react for five minutes. A second test tube with 5(mL) sample without reagents (blank) was prepared. Then the two test tubes were used with the color wheel to determine the nitrite N concentration Laboratory Water Quality Determination Water quality determination conducted in the laboratory included solids, TKN, and nitrate N, with procedures and material requires as listed below. Total solids(ts), volatile solids(vs), and total non volatile solids(tnvs) Materials needed are ceramic crucibles, centrifuge, 105 C oven, and 500 C oven. To exclude soluble solids (like salts), the sample was centrifuged to divide total volatile solids from soluble solids. First one crucible was weighted. Then 90(mL) of raw sample were centrifuged, the supernatant was disposed, the precipitant was re suspended in DI water, filled into the crucible and dried for 24 hours in the 105 C oven, and then weighted again. Finally the crucible with dried sample was burned for 2 hours in the Page 29

52 Methods 500 C oven and weighted again. Total Solids, non volatile solids and volatile solids were calculated using equation (7). (7) Total Kjeldahl Nitrogen (TKN) Organic nitrogen and ammonia was determined together and is referred to as Total Kjeldahl Nitrogen. A digestion apparatus for 100mL Kjeldahl flasks and a distillation apparatus, equipped with a steam generating vessel was used. Samples for measurement of soluble TKN were centrifuged and filtered (supernatant was used) and the raw sample was used to determine total TKN. The Procedure is listed below. Standards & Blanks Blanks: 20mL of DI water. Standards: 5mL of (NH 4 ) 2 SO 4 (100 mg/l) diluted with DI water into 100mL flask. Reagents Digestive Mixture; 100g of granular K 2 SO 4 (Potassium Sulfate), 10g of CuSO 4 (Copper Sulfate anhydrous powder), 1g of selenium to a mortar. The mixture was grind until fine and was mixes well. Boric Acid Indicator Solution (1 L): 20g of pure boric acid was dissolved in 700ml of hot water and the cooled solution was transferred to a 1 liter volumetric flask Page 30

53 Methods containing 200 ml of ethanol and 20mL of mixed indicator solution. After mixing, 0.05N NaOH (Sodium Hydroxide) were added until a color change from pink to pale green was detectable when 1mL of the solution was treated with 1mL of water. The volume was brought up with distilled water and mixed thoroughly. Mixed Indicator Solution: 0.330g Bromcresol Green and 0.165g Methyl Red were dissolved in 500mL of Ethanol. Sodium Hydroxide (NaOH): 625g of solid Sodium Hydroxide was dissolved in 800mL of distilled water in a 1 liter flask; (30% Solution for Ammonia Samples) 300g into 1000mL. Procedure 1. Samples (Samples analysised in duplicate) The sample was homogenized in a blender, poured into a plastic container, foam was removed and returned into its original container. The sample was poured into a beaker containing a stir bar and placed on a stir plate. 5 ml were filled into a 200 ml flask and bring to volume with DI water. The sample was poured into a beaker containing a stir bar and 20 ml of sample was filled into the digestion tubes. 2. For Soluble TKN The sample was shaken and then poured into six 30 ml centrifuge tubes (total 180 ml) and centrifuged at rpm for 5 minutes. liquid was poured into new Page 31

54 Methods sample bottles and the solids were discarded using DI water to rinse the centrifuge tubes. 3. For Particulate TKN 4. The sample was filtered using a glass fiber pre filter and poured back into the bottle. The sample was filtered again using a 0.45 micro filters. 5. Digestion The temperature was adjusted to 160 C for 1 hour. The total cycle temperature was set on 450 C and total cycle time 2 4 hours (If the liquid has not been clear, it was continued to digest). 1 scoop of digestive mix was added to the glass tubes. The sample was shaken and poured into a plastic container containing a stir bar, and then the stir plate was turned on. 20 ml of sample were filled into a labeled TKN test tube and 3 ml of concentrated Sulfuric Acid (H 2 SO 4 ) were added. The samples were placed on the digestion unit (If the liquid has not been clear, it was continued to digest). 6. Distillation and Titration 20 ml of DI water were added to the digested samples, mixed, and transferred into a Kjeldahl flask. 5 ml of boric acid indicating solution was filled into an Erlenmeyer flask and placed under the collection spout. The Kjeldahl flask was connected to the distillation unit and 10 ml of the NaOH solution (Sodium Hydroxide) were added. NOTE: for Ammonia samples 30% NaOH solution were used. 30 ml of distillate were collected, a stir bar was added, and placed on a stir Page 32

55 Methods plate. Using 0.007N H 2 SO 4 (Sulfuric Acid) the sample was titrated until a permanent pale pink. 7. Calculations (8) NO 3 N (Nitrate) Materials Spectrophotometer set to read Transmission at 543 nm NO 3 Color Reagent Diluted NH 4 CL EDTA standard Standards: KNO 3 solution (10 mg/l as Nitrogen) 0.1(mg/L): 1mL into a 100ml volumetric bring to volume with DI water 0.5(mg/L): 5mL into a 100ml volumetric bring to volume with DI water 1.0(mg/L): 10mL into a 100mL volumetric bring to volume with DI water 25 ml of standard was poured into a 100ml beaker and filled up to volume with diluted NH 4 CL EDTA. Procedure 25mL of sample were filled into a 100mL volumetric flask, and filled up to volume with diluted NH 4 CL EDTA and poured into a beaker. Page 33

56 Methods The sample was filled into the column. A beaker was placed under the column and the sample collected. 2mL of color reagent were added to the beaker containing the sample and swirled; the sample was allowed to sit for 10 minutes and filled into specified cuvettes. The spectrophotometer was used with the cuvettes and the absorption at 543 nm was taken. The calibration formula is shown in equation (9) (9) Samples with a nitrate concentration above the limit of quantization were diluted until the measurement was inside the limits. To determine the nitrate concentration the value was multiplied by the dilution factor. Page 34

57 Methods 3.3 Operational Procedures Operational procedures included shrimp stocking, harvest, shrimp feeding, water circulation and ph control Shrimp Stocking and Harvest Approximately 1,050,000 PL 8/9 shrimp were stocked on May 2007 (350,000 animals per unit). Each unit had a surface area of 250m², yielding an initial stocking density of 1400(shrimp/m²). The animals were monitored and acclimated for 24 hours in 800gal (3028 L) tanks, placed at the front of each unit. The tanks were supplied with air through porous air diffusers from a 1.5hp (1,118 W) regenerative centrifugal blower. The air supply was also mixing the system, thereby reducing settling in the tanks. The initial salinity in the tanks was 30(g/L), while system salinity was 10(g/L). The animals were acclimated to the new salinity at a rate of 1.5 to 2 (g/l hr), using the following mixing mass balance equation (Kirk, 2004): 0 (10) Q represents the flow rate in [gal/min] needed for the given time interval, V represents the constant volume of the tank in [gal], represents the time interval for the given flow rate, represents the initial salinity in [g/l] represents the final salinity in [g/l] and the salinity of the water that is being added from the unit in [g/l]. Page 35

58 Methods At the end of a 149 day growing season, units were individually harvested. The animals were captured with a seine drawn through the units. This was repeated until 99% of the animals were harvested Shrimp Feeding The feeding procedure was adopted from Kirk, Therefore feed was uniformly broadcast to the units, the amount fed based on percent body weight (from 30% to 2% body weight per day). The feed contained 32% protein presented as a 3/32 inch pellet and applied 3 times per day. Feed application was also adjusted in response to system water quality (Table 4). The bottoms of the units were sampled with a net to certain accumulation of uneaten feed as a result of overfeeding, at which point, the feeding rate would be adjusted accordingly. Table 4: Critical water quality criteria for food application. Parameter Value Feed rate Ammonia >3.0 [mg/l] Cut feed by half Nitrite >2 [mg/l] Cut feed by half Nitrite >2.5 [mg/l] Stop feeding DO <3 [mg/l] Cut feed or stop feeding PH Control The ph control procedure was adopted from Kirk, Therefore the ph in the units was controlled using CO 2 injection via diffusers and if needed late in the season with NaHCO 3 supplementation. The flow rate of CO 2 into the units was maintained constant Page 36

59 Methods throughout the season. Interval timers linked to normally closed solenoid valves were adjusted as needed to maintain ph in each of the units at or below about 8.0 (Table 5). Table 5: Critical water quality criteria for ph control. Parameter Value CO 2 Intake ph >8.0 Add CO 2 for 30 minutes ph >8.2 Add CO 2 for 60 minutes ph >8.5 Add CO 2 for 120 minutes alkalinity <100 [mg/l] Add 50 lb of NaHCO 3 To raise the ph if needed NaHCO 3 was added to each of the units. Therefore the alkalinity was measured and if the alkalinity was below 100 (mg/l), 50lb (24,250g) NaHCO 3 were added Water Exchange Water exchange between shrimp units, tilapia zone, settling tank/denitrification reactor, and/or nitrification reactor was adjusted based on desired selection of hydraulic detention in shrimp units or adjusted according to water quality needs to sustain shrimp health Water exchange rates were measured once per week, timers recorded accumulated pumping times for each unit. The circuit diagram of the water circulation control system box is shown in Figure 15. Page 37

60 Methods Figure 15: Water exchange control circuit diagram. Source: Own drawing, design by Kendall Kirk. Page 38

61 Methods Sequencing Batch Reactor (SBR) Control During the last 2 months of the season the SBR was operated. The SBR sequence is given in Table 6. Table 6: SBR cycle settings (times) Stage Time Action Fill 60 min Aerating, valve closed Mix 20 min Aerating, valve closed Settle 20 min No aerating, valve closed Decant 20 min No aerating, valve opend The nitrification reactor decant (or discharge) valve was opened and with an electrically operated actuator with the position controlled by limit switches and timers or high level float switches providing for water storage with gravitationally driven flow back to the shrimp units. The sequencing of the reactor was controlled by an electrical circuit shown in Figure 16. Page 39

62 Methods Figure 16: SBR control circuit diagram. Source: Own drawing, design by Kendall Kirk. Page 40

63 Methods 3.4 Data Analysis and Manipulation Interpolations Certain parameter (such as Nitrate, alkalinity, photosynthesis and solids) were measured weekly. To combine these system representations with daily values, weekly representation of system behavior were linearly interpolated to yield daily representations of that parameter. The calculation is shown in equation (11). (11) Re carbonation Rate Inorganic carbon supplementation to the system from added CO2 through diffusers was calculated with the method described in Kirk, This calculation provided system recarbonation rate as [mmol/l (hr CO 2 addition)] Day and 7 Day Moving Averages The 7 day averages were calculated for each of the water quality measurements (temperature, DO, salinity, TAN, nitrite concentration and Secchi depth) and feed rate. 2 day averages were calculated for observed rates in the system, if possible (ammonia transformation). The averages were calculated by averaging the measurements for the previous 3.5 days and those for the following 3.5 days (previous one day and following one day respectively). Page 41

64 Methods Unionized Ammonia Unionized ammonia was calculated using equation (12). (12) Where [NH 3 N] represents the unionized ammonia concentration (mg N/L), [TAN] represents the total ammonia nitrogen concentration (mg N/L), and T represents temperature in C. (Kirk,2004) Carbon Mass Balance Equations This chapter includes all calculations made to develop carbon mass balances from the raw data. The unit of every calculated rate is transferred to. Assumptions and facts are given to each formula and eventually the different rates were put together to derive one carbon mass balance. Carbon Input from Feed Application The carbon input rate due to feed application was calculated using equation (13). (13) Where is the total amount of feed in [g] applied during the time frame in [day]. representing the shrimp culture area in. It is assumed, that the applied feed contains 90% volatile solids (VS) and 50% of VS is carbon. The organic carbon input rate from feed applicaiton is calculated as follows: Page 42

65 Methods (14) All parameters are previously defined. CO 2 Supply The recarbonation rate from CO 2 addition used is as previously presented in KIRK, This rate is converted to carbon input using ² equation (15) (15) Where is the molar based volumetric re carbonation rate, the molecular weight of carbon is 0.012, 2 is the accumulated time that CO 2 injection was performed in [hr] during the time interval in [day]. is the volume of the unit that CO 2 was applied to in [L]. All other parameters were previously defined. Inorganic Carbon Input from NaHCO 3 Supplementation The carbon input rate due to sodium bicarbonate supplementation was ² calculated based upon molecular weights of carbon and sodium bicarbonate as shown in equation (16) (16) Page 43

66 Methods Where 3 is the total amount of sodium bicarbonate added in [g] during the time interval in [day]. The molecular weight of carbon is 12 and of Sodium bicarbonate 84. All other parameters were previously defined. Algal Carbon Fixation The rate of algal carbon fixation is calculated based on the volumetric ² 24hr photosynthesis rate. The net photosynthesis rate on a per unit area base was calculated using equation (17) (17) Where is the volumetric 24hr photosynthesis rate in, V the volume [L] and A the area in[m²] of the shrimp culture footprint. The photosynthetic rate is represented as (KING, 1976); (18) Assuming the molecular ratio of oxygen production to carbon fixation is 1:1. Therefore algal carbon fixation is calculated using: ² (19) With the molecular weight of carbon of 12 and of oxygen gas of 32. All other parameters were previously defined. Page 44

67 Methods Carbon Storage in Sludge Accumulation The rate of carbon storage due to sludge accumulation in the settling tank/denitrification reactor was calculated based on the volatile solids ² content of the sludge (20) Where the sludge volume V is given in [L] and the volatile solids concentration VS in. It is assumed that 50% of the sludge VS is carbon, the per unit area based rate of carbon storage by sludge accumulation was calculated using equation (21) ² (21) where all parameters are previously defined. Shrimp Biomass Carbon Content The rate of carbon accumulation in shrimp biomass was calculated ² based on the weight difference of the shrimp during an observed time interval. The shrimp wet weight was measured to contain 80% water, and 50% of the shrimp dry weight is assumed to carbon, The carbon content of shrimp can be calculated with equation (22) (22) Page 45

68 Methods Were is the total shrimp wet weight in [g] gained during the time interval in [day]. The rate of carbon gain by shrimp biomass growth per unit area is calculated using equation (23). 0.1 ² (23) With all parameters previously defined CO 2 Outgassing The rate of carbon output due to CO 2 outgassing is calculated based on ² the assumption that there are no other appreciable carbon outputs from the system. Since the sum of all inputs to the system at steady state must be equal to all outputs plus accumulation, the carbon removal rate due to CO 2 outgassing can be determined from a total carbon mass balance. The total carbon mass balance at steady state can be calculated as in equation (24) shown. 0 (24) Were all parameters are previously defined. The rate of carbon output due to outgassing is calculated using equation (25). ² (25) Were all parameters are previously defined. Page 46

69 Methods Nitrogen Mass Balance Equations System nitrogen mass balances were developed for shrimp culture units, for individual reactors and for the system at Nitrogen transformation are expressed as. Nitrogen Input from Feed Application The rate of nitrogen input from feed application is based on the ² protein content of feed. The feed protein content was 32% and the assumption was made that protein contains 16% nitrogen. Therefore the nitrogen input due to feed application can be calculated using equation (26) (26) Were all parameters are previously defined. The transformation of N mass per unit area based rate was made using equation (27) ² (27) With all parameters previously defined. Algal Nitrogen Fixation The rate of algal nitrogen fixation was calculated using an previously ² measured algal C:N ratio of 5.6:1 (BRUNE et al., 2003). Therefore equation (28) may be used to calculate the rate of algal nitrogen fixation ² (28) Page 47

70 Methods With all parameters previously defined. Nitrogen Storage due to Sludge Accumulation The rate of nitrogen storage due to sludge accumulation was ² calculated using previously measured C:N ratios of activated sludge of 6.5:1 (BRUNE et al., 2003). Therefore equation (29) was used to calculate the rate of nitrogen storage due to sludge accumulation ² (29) With all parameters previously defined. Nitrogen Storage from Shrimp Growth The rate of nitrogen storage resulting from shrimp growth was ² calculated using the determined shrimp biomass C:N ratio of 4.17:1. Therefore equation (30) was used to calculate the rate of nitrogen storage resulting from shrimp growth ² (30) With all parameters previously defined. Nitrification and Denitrification The rate of nitrogen removal by nitrification and denitrification was ² calculated based on the total nitrogen mass balance, showed in (31). 0 (31) Were all parameters are previously defined. Page 48

71 Methods The rate of nitrogen output due to nitrification and denitrification is calculated using equation (32). (32) Were all parameters are previously defined. Page 49

72 Methods Shrimp Biomass Estimation Direct measurements of shrimp numbers or biomass were not possible. Consequently the feed consumption is used to estimate the shrimp biomass and shrimp numbers throughout the season. Wyk (2000) provided shrimp feed uptake vs. body weight at optimal conditions. This relationship is presented in Figure 17. Feed intake, (% of average body weight) y = 10.55x 0.45 R² = Feed Conversion Power (Feed Conversion) Average shrimp body weight, (g) Figure 17: Shrimp feed intake in % of average body weight dependent of their average body weight at optimum growth conditions. Source: Data from Wyk, Therefore equation (34) was used to estimate shrimp biomass in the PAS. % Therefore: % 100 % (33) (34) Page 50

73 Results and Discussion 4 RESULTS AND DISCUSSION 4.1 Obtained Seasonal Data Outline To better understand the raw and manipulated data, bracketed time frames and photos are used to isolate and emphasize significant events during the season and are combined with the data presentation in appendix K J. The growth season in 2007 was different from previous seasons, especially in terms of non uniformly of experimental execution, and in resulting process response. In 2007 algal production, nitrification and denitrification was combined. In addition, the maximum feed application was driven to the edge of system capacity and unplanned events forced changes in the operational procedures. However, these changes led to the possibility to separate the grow out season into different experiments for a better understanding of the maximum capacity of the system. To define these different experiments, an event history was generated for each of the units. At the beginning of each data presentation, a timeline and the general condition of the unit is shown. Selected events are defined from time of stocking, illustrated at the bottom of page as a point of reference. Feed input, macro/micro visualization, water circulation, temperature, dissolved oxygen, salinity, ph, secchi depth, volatile solids, alkalinity, total ammonia nitrogen, Page 51

74 Results and Discussion unionized ammonia, nitrite, nitrate, total Kjeldahl nitrogen, photosynthesis and shrimp production is presented vs. days from stocking, or actual date as shown. Vertical dashed lines represent distinct periods and lines represent times spans used to generate moving averages of the parameter of interest. Page 52

75 Results and Discussion Water Quality Summary The data summarized in this section is found in the appendix E to J. The temperature trends are similar for each of the units. During the early and mid season the temperature oscillated between 26 C and 32 C. During the end of the season the temperature gradually fell to 22 C to 28 C. The water temperature of the nitrification reactor tends to be lower, which came from the fact that the nitrification reactor was not covered by the greenhouse. Temperatures ranged from 26 C to 31 C in mid season and 18 C to 25 C at the end of the season. In the beginning of the season, high DO fluctuations resulted from excessive algal productivity and are proportional to algal biomass and photosynthetic available radiation energy. In mid July two 0.75 hp fountain aerators in each unit were installed to support night time respiration demands of the growing shrimp. Until day 103 the DO average was above 4. After that date the system changed into a nitrification dominated system and up to six 0.75 hp fountain aerators in each unit supported the respiration requirements. By aerating the DO average was kept above 3 with extremes as low as 1.9. During that timeframe unit #2 was exchanging with the nitrification reactor and due to lower solids concentration the DO in this unit was kept at an average of above 4. At the end of the season, when temperatures went down, the DO average went up to about 5 to 6. Page 53

76 Results and Discussion The salinity of the units throughout the season was stable and averaged around 11 for every unit. The sharp rise around day 105 was the result of salt addition to protect the shrimp from nitrite toxicity. Other fluctuations came from different water exchange with unit #3, which was not salted up around day 105. The ph in each unit during the early and mid season ranged from 7.6 to 8.5 and at the end of the season around 7 to 7.5. The ph was artificially controlled during the first and the last third of the season. At high algal productivity and low respiration the ph had to be lowered by CO 2 intake. In mid season, CO 2 production through respiration was high enough to meet algal uptake needs and the ph ranged around 7 to 8. At the end of the season, high alkalinity destruction by means of nitrification, without alkalinity generation through mineralization in the denitrification reactor, the ph in unit #1 and #4 was held up at levels around 7 to 7.5 by adding NaHCO 3. Unit #2 however was exchanging with the nitrification reactor and the denitrification reactor/settling tank and no artificial NaHCO 3.had to be added. In general the TAN concentration averaged between 1 and 2. Due to excessive mixing of the denitrification reactor/settling tank two major peaks of ammonia appeared on day 34 and day 101. The first peak had maximal ammonia concentrations of up to 5 in unit #1, #2 and #4 and up to 9 in unit #3. During the second peak maximum ammonia concentrations of 8 in unit #1, #2 and #4, 24 in unit #3, 44 in the denitrification reactor and 38 in the nitrification reactor were Page 54

77 Results and Discussion measured. Following smaller peaks of TAN concentrations in unit #3 resulted from water exchange between the denitrification reactor and unit #3. In the beginning of the season high ph and high temperatures led to high unionized ammonia concentrations, with peaks of 0.5 in unit #1, #2 and #4. During the season, with lower temperatures and lower ph, the NH 3 N concentration fell below 0.1. In unit #3 maximum NH 3 N concentrations of 1.3 were seen. Nitrite appeared in the system after the first excessive mixing of the denitrification reactor/settling tank with maximum concentrations of 5 in unit #1 to #4. In mid season two main peaks of nitrite were seen in every unit, with maximum concentrations of 3 to 4. After the second excessive mixing of the denitrification reactor/settling tank, nitrite concentrations went up to 8.5 in unit #1, #2 and #4 and up to 14 in unit #3. After the units turned bacterial dominating on day 101, nitrite in units #1 and #4 almost disappeared, with one peak at the very end of the season. In unit #2, since exchange through the nitrification reactor, the denitrification reactor/settling tank, and unit #3 was enabled at the end of the season, several nitrite peaks occurred. Nitrate concentrations of the three shrimp units were measured weekly. The concentration profiles reflect the flow through the nitrification/denitrification reactors. After the mixing of the denitrification reactor on day 101 the units became bacterial dominated and NO 3 N started to build up in the units. On day 119, unit #2 began to exchange with the denitrification reactor/settling tank and nitrate began to fall until the Page 55

78 Results and Discussion exchange in unit #2 was stopped. When unit #1 and #4 started to exchange with the denitrification reactor and unit #3 nitrate concentrations fell also. Particulate solids in unit #1, #2 and #4 averaged between 200 and 300, whereas no substantial difference in solids concentrations due to different retention times could be seen. Slightly lower solids concentrations were measured in unit #3 due to Tilapia harvest and solids settling in the denitrification reactor/settling tank. After day 101, when unit #1 and #4 were isolated solids concentrations went up to 900 and 700 respectively. Since unit #2 was exchanging with the reactor after day 119, solids concentrations ranged between 300 and 400. A total of 23,400 (20,800 ) was harvested at the end of the season (149 days from PL 8 shrimp to an average of harvest weight of 11.3 ). The average feed rate throughout the season was to the shrimp. With peak feed rates of (1645 (649 ). ) was fed Page 56

79 Results and Discussion System Configuration and Operational Adjustments Mass balances for the total season are not revealing as a result of major alterations in system configuration and operation. The system operation and water exchange was done in response to major unexpected system events, or in an attempt to optimize the overall reactor performance. As Figure 35 to Figure 126 (appendix K J) show, there were times of algal dominance of nitrogen processing, and other times of nearly complete nitrification dominance and in unit #2, a combination of these two processes. Within the event timelines selected snap shots were defined to describe the system performance. Individual mass balances are calculated and presented for these times frames. These summaries are based on, flow rates, system configuration, steady state behavior, volatile solids concentration, water quality, and excessive mixing events of the denitrification reactor/settling tank. These individual snap shots are described in Table 7. Page 57

80 Results and Discussion Table 7: Characterization of 2007 PAS component system performance. Unit/reactor Dates Main characteristic Unit #1 to # to (Day 43 through Day 96) Algal dominated; Denitrification reactor on line; Nitrification reactor on line; Tilapia confinement on line. Unit # to (Day 116 through Day 137) Unit # to (Day 116 through Day 137) Unit # to (Day 116 through Day 137) In situ Nitrification, no exchange. Algal supplemented; Denitrification reactor on line; Nitrification reactor on line; Tilapia confinement on line. In situ Nitrification, no exchange. Nitrification Reactor to (Day 43 through Day 96) to (Day 122 through Day 140) + separate measurements Ammonia and nitrite oxidation (Nitrification). Denitrification reactor to (Day 129 through Day 137) Nitrate reduction (Denitrification). Page 58

81 Results and Discussion 4.2 Photoautotrophic Dominated PAS Performance From day 1 to day 110 all units were algal (photoautotrophic) dominated. A representative timeframe, approximating steady state behavior, was defined from day 43 through day 96. This timeframe lies between two excessive mixing events in the denitrification reactor/settling tank on day 34 and 101, and their consequences, high shrimp mortality, unstable water quality, interrupted water exchange, and non steady state behavior. Nitrogen and carbon mass balances around the three shrimp units were constructed and are presented. At the beginning of each mass balance a water quality summary is presented, with average parameters for the observed timeframe. Finally, the performance of the three units is compared, with major differences discussed, and graphically presented. Average water quality parameters are presented in Table 8, Table 11 and Table 14 for the units #1, #2, and #4, including photosynthesis and feed input rates. During the season the average shrimp weight was measured weekly. The shrimp weight difference and the calculated total shrimp biomass increases are shown in Table 9, Table 12, and Table 15. Since the units were exchanging with the denitrification reactor/settling tank combo, the nitrification reactor and the tilapia confined unit, mass fluxes had to be taken into account and flow rates are shown in Table 10, Table 13, and Table 16. Calculated nitrogen and carbon mass balances for the units #1, #2, and #4 from day 43 to 96 are presented in Table 17 to Table 22. TAN, Nitrite, Nitrate and total carbonate Page 59

82 Results and Discussion concentrations were essentially the same throughout this time period and slight differences between in and outflow concentrations of these parameters were negligible. Solids removal trough denitrification reactor/settling tank and the tilapia confined unit #3 was considerable. Therefore, the only carbon inputs were feed application and CO 2 addition. The carbon outputs were measured solids removal, CO 2 outgassing and shrimp output. The only input of nitrogen was by feed application and the outputs were observed solids removal and shrimp output. Table 8: Average water quality and system parameters; Unit #1; day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg C/L) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 11, ,000 Page 60

83 Results and Discussion Table 9: Shrimp biomass and individual weights; Unit #1 day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Difference Day 43 Day 96 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 644,983 65, ,810 Table 10: Water flows and detention times; Unit #1, day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) 76, ,439 Retention time (day) Water exchanges (#/day) Table 11: Average water quality and system parameters; Unit #2; day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg O 2 /L/day) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 11, ,000.0 Page 61

84 Results and Discussion Table 12: Shrimp biomass and individual weights; Unit #2 day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Difference Day 43 Day 96 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 874,188 70, ,027 Table 13: Water flows and detention times; Unit #2, day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) 194,797 6, ,098 Retention time (day) Water exchanges (#/day) Table 14: Average water quality and system parameters; Unit #4; day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg C/L) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 10, ,000.0 Page 62

85 Results and Discussion Table 15: Shrimp biomass and individual weights; Unit #4 day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Difference Day 43 Day 96 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 637, , ,841 Table 16: Water flows and detention times; Unit #4, day 43 through day 96 (Photoautotrophic dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) 150, ,576 Retention time (day) Water exchanges (#/day) Table 17: Carbon mass balance summary; Unit #1; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Algal fixation r CSA Measured solids removal r CSS Shrimp output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input , , , , , Page 63

86 Results and Discussion Table 18: Nitrogen mass balance summary; Unit #1; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r NIF Algal fixation r NSA Measured solids removal r NSS Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input Table 19: Carbon mass balance summary; Unit #2; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Algal fixation r CSA Measured solids removal r CSS Shrimp output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input , , , , Page 64

87 Results and Discussion Table 20: Nitrogen mass balance summary; Unit #2; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r NIF Algal fixation r NSA measured solids removal r NSS Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input Table 21: Carbon mass balance summary; Unit #4; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Algal fixation r CSA Measured solids removal r CSS Shrimp output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input , , , Page 65

88 Results and Discussion Table 22: Nitrogen mass balance summary; Unit #4; day 43 through day 96 (Photoautotrophic dominated). Parameter Feed input r NIF Algal fixation r NSA Measured solids removal r NSS Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input From day 43 to 96 hydraulic detention times ranged from 0.5/day in unit 1, 1.3/day in unit #2 and 1.0/day in unit #4. Volatile solids concentration range, between the 3 shrimp units, was 277 ±20 and the photosynthesis range was 59.4 ±6, additionally no trend was seen, that indicated a influence of the detention time on solids concentration, water quality, or photosynthesis. Also, the nitrogen processing in each unit was dominated by photosynthesis and therefore little differences in rates or overall mass balances were observed. Therefore the three mass balances have been pooled to yield a single averaged carbon and nitrogen mass balance illustrated in Figure 18, and Figure 19. Page 66

89 Results and Discussion Feed Input [g C/m² day] 99.6% of Input Water Column Algal Fixation [g C/m² day] 34.1% of Input Recarbonation 0.16 [g C/m² day] 0.4% of Input Measured Solids Removal 6.64 [g C/m² day] 16.9% of Input Unaccounted C Removal 29.1 [g C/m² day] 74.1% of Input Shrimp Output 3.45 [g C/m² day] 9% of Input Figure 18: Carbon mass balance for the photoautotrophic dominated units, day 43 through day 96. The total carbon input of the photoautotrophic dominated units averaged Shrimp output accounted for 9% of the carbon removal ² (3.45 ² ). 16.9% (6.64 ) of the carbon was removed from the shrimp ² units through unit #3 by settling and tilapia feeding (measured solids removal) % (29.1 ² ) of carbon was unaccounted for, dominated by CO 2 outgassing from the system. Page 67

90 Results and Discussion Feed Input 4.45 [g N/m² day] 100% of Input Water Column Algal Fixation 2.39 [g N/m² day] 53.7% of Input Observed Solids Removal 1.13 [g N/m² day] 25.3% of Input Unaccounted N Removal 2.48 [g N/m² day] 55.8% of Input Shrimp Output 0.85 [g N/m² day] 19% of Input Figure 19: Nitrogen mass balance for the photoautotrophic dominated units, day 43 through day 96. Feed application accounted for 100% of the Nitrogen input (4.45 ). Algal ² productivity accounted for nitrogen fixation of 2.39 ) (53.7% of feed input), ² and 1.13 (25.3% of feed input) was removed through unit #3 by settling and ² tilapia uptake. However, 2.48 (55.8% of feed input) of nitrogen removed, ² was not accounted for in direct field measurements. Outgassing of NH 3 was not significant due to low ph in the system, also ammonia, nitrite and nitrate concentrations were constant during this time period. Since nitrate did not accumulate at this time, and nitrogen content of the nitrifying biomass was not significant throughout this time period, nitrogen removal by nitrification is assumed to be insignificant. The only other Page 68

91 Results and Discussion sink of nitrogen, not measured, was in the settling and denitrification reactor (unit #3C and #3A). Due to inefficient food processing by shrimp, uneaten feed most likely exited the system through water exchange with unit #3. In fact the two mixing events occurring in the settling tank (unit #3A) on day 34 and day 101 released large amounts of nitrogen into the water column, which came from previous settling of algal biomass, uneaten food and shrimp fecal matter. The capacity of nitrogen processing with phototrophic nitrogen fixation is dependent upon the degree that solids that can be removed from the water column, and therefore on the maintenance of a low algal cell age. At an average carbon ² fixation, yielding an algae production of 44.5 at an average observed particulate solids concentration of 278 suggests an average algae cell age of 6.2 days. System capacity of 4.45 of nitrogen input at hydraulic detention times ² of 0.5/day to 1.3/day with adequate solids removal was demonstrated from day 43 to day 96. Page 69

92 Results and Discussion 4.3 Chemoautotrophic Dominated PAS Performance Mixing in the denitrification reactor/settling tank, unit #3A, on day 101, led to release of previously stored nitrogen in excess of algal processing capacity leading to high ammonia concentrations of up to 8. Therefore exchange of all units through the denitrification reactor/settling tank, the tilapia confined unit, and the nitrification reactor was suspended on day 104. Without settling/harvesting of algal biomass and with high water column ammonia concentrations, nitrifying bacteria became competitive; As a result, within one week the ammonia previously fixed by algae was being processed through nitrification. Units #1 and #4 were maintained without exchange (as batch operated units) from day 104 to day 138 and from day 104 to day 140 respectively. Exchange in unit #2 was restarted on day 116, when ammonia concentrations in unit #3 dropped to 3. In addition the performance of the nitrification reactor was improved by redesigning it as a sequencing batch rector and it was operated this way for the duration of the season. This design allowed keeping nitrifying biomass in the nitrification reactor water column. Average water quality parameters are given in Table 23, Table 26, and Table 29 for the units #1, 2#, and #4, including photosynthesis and feed input rates. During the season the average shrimp weight was measured weekly. The shrimp weight difference and the calculated total shrimp biomass increases are shown in Table 24, Table 27, and Table 30. Since unit #2 was exchanging with the denitrification reactor/settling tank, the Page 70

93 Results and Discussion nitrification reactor and the tilapia confined unit, mass fluxes had to be taken into account and flow rates are shown in Table 28. Calculated nitrogen and carbon mass balances for the units #1, #2, and #4 from day 116 to 137 are presented in Table 32 to Table 37. Table 23: Average water quality and system parameters; Unit #1; day 116 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg O 2 /L/day) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 18, , ,000.0 Table 24: Shrimp biomass and individual weights; Unit #1 day 116 through day 137 (Bacterial dominated). Parameter Unit Difference Day 116 Day 137 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 118, , ,145 Page 71

94 Results and Discussion Table 25: Water flows and detention times; Unit #1, day 116 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) Retention time (day) inf inf Inf Water exchanges (#/day) Table 26: Average water quality and system parameters; Unit #2; day 126 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg O 2 /L/day) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 17, ,500 24,000.0 Table 27: Shrimp biomass and individual weights; Unit #2 day 126 through day 137 (Bacterial dominated). Parameter Unit Difference Day 126 Day 137 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 311, ,053, ,341.5 Page 72

95 Results and Discussion Table 28: Water flows and detention times; Unit #2, day 126 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) 69, ,940.8 Retention time (day) Water exchanges (#/day) Table 29: Average water quality and system parameters; Unit #4; day 116 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Temperature ( C) DO (mg/l) Salinity (g/l) ph ( ) Particulate Solids (mg/l) Secchi depth (cm) depth (cm) TAN (mg N/L) NH 3 (mg N/L) NO 2 (mg N/L) NO 3 (mg N/L) Total carbonate carbon (mg O 2 /L/day) Alkalinity (meq/l) NET photosynthesis (mg O 2 /L/day) Respiration (mg O 2 /L/day) Daily feed rate (lb C/ac/day) Daily feed rate (g C/day) 13, ,000.0 Table 30: Shrimp biomass and individual weights; Unit #4 day 116 through day 137 (Bacterial dominated). Parameter Unit Difference Day 116 Day 137 Average shrimp weight (g) Shrimp count (#/m²) Total shrimp biomass (g/unit) 149, , ,083.3 Page 73

96 Results and Discussion Table 31: Water flows and detention times; Unit #4, day 116 through day 137 (Bacterial dominated). Parameter Unit Average Minimum Maximum Flow out (L/day) Retention time (day) inf inf Inf Water exchanges (#/day) Table 32: Carbon mass balance summary; Unit #1; day 116 through day 137 (Bacterial dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Carbonate accumulation r CSC Algal fixation r CSA Measured solids accumulation r CSS Shrimp output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input Page 74

97 Results and Discussion Table 33: Nitrogen mass balance summary; Unit #1; day 116 through day 137 (Bacterial dominated). Parameter Feed input r NIF Algal fixation r NSA Measured solids accumulation r NSS Nitrate accumulation r NSN Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input Table 34: Carbon mass balance summary; Unit #2; day 126 through day 137 (Bacterial dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Carbonate accumulation r CSC Algal fixation r CSA Observed solids removal r CSS Shrimp Output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input Page 75

98 Results and Discussion Table 35: Nitrogen mass balance summary; Unit #2; day 126 through day 137 (Bacterial dominated). Parameter Feed input r NIF Algal fixation r NSA Measured solids removal r NSS Nitrate accumulation r NSN Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input N/A N/A N/A N/A N/A Table 36: Carbon mass balance summary; Unit #4; day 116 through day 137 (Bacterial dominated). Parameter Feed input r CIF CO 2 input r CIR Carbonate input r CIC Carbonate accumulation r CSC Algal fixation r CSA Observed solids accumulation r CSS Shrimp output r COS CO 2 outgassing r COC Ave. Min. Max. Total Δ % of Total (g C/m²/day) (g C/m²/day) (g C/m²/day) (g C/m²) input Page 76

99 Results and Discussion Table 37: Carbon mass balance summary; Unit #4; day 116 through day 137 (Bacterial dominated). Parameter Feed input r NIF Algal fixation r NSA Measured solids accumulation r NSS Nitrate accumulation r NSN Shrimp output r NOS Unaccounted r NON Ave. Min. Max. Total Δ % of Total (g N/m²/day) (g N/m²/day) (g N/m²/day) (g N/m²) input During this period TAN, Nitrite, and total carbonate concentrations were stable in unit #1 and #4, but since unit #1 and #4 were operating without exchange through the denitrification reactor, NaHCO 3 had to be added to maintain alkalinity levels. Therefore the carbon input was feed application and NaHCO 3 supplementation. The major output of carbon was CO 2 outgassing. The only nitrogen input was from feed application and the major outputs of nitrogen were from through nitrification seen as accumulating nitrate concentrations. These mass balances for unit #1 and #4 are summarized in Figure 20 and Figure 21, and the mass balances for unit #2 summarized in Figure 22 and Figure 23. Page 77

100 Results and Discussion Feed Input Unit # [g C/m² day] 96.54% of Input Unit # [g C/m² day] 97.68% of Input CO 2 Outgassing Unit # [g C/m² day] 90.83% of Input Unit # [g C/m² day] % of Input Water Column Observed Solids Accumulation Unit # [g C/m² day] 10.13% of Input Unit # [g C/m² day] 7.11% of Input CO 3 Input Unit #1 2.3[g C/m² day] 3.46% of Input Unit #4 1.2 [g C/m² day] 2.32 % of Input Shrimp Output Unit #1 1.4 [g C/m² day] 2.06% of Input Unit #4 1.9 [g C/m² day] 3.8% of Input Figure 20: Carbon mass balance; Chemoautotrophic dominated, units #1 and #4, day 116 through day 137. Page 78

101 Results and Discussion Feed input Unit #1 7.4 [g N/m² day] 100% of Input Unit #4 5.6 [g N/m² day] 100% of Input Water Column Observed solids accumulation Unit # [g N/m² day] 14.19% of Input Unit # [g N/m² day] 9.84% of Input Shrimp output Unit #1 0.3 [g N/m² day] 4.47% of Input Unit #4 0.5 [g N/m² day] 8.14% of Input Nitrate accumulation Unit # [g N/m² day] 43.69% of Input Unit # [g N/m² day] 34.72% of Input Unaccounted organic solids accumulation Unit # [g N/m² day] 30.1% of Input Unit # [g N/m² day] 15.5% of Input Denitrification Unit #1 1.6 [g N/m² day] 21.6% of Input Unit #4 2.6 [g N/m² day] 46.5% of Input Figure 21: Nitrogen mass balance; Chemoautotrophic dominated, units #1 and #4, day 116 through day 137. Page 79

102 Results and Discussion Feed Input 61.7 [g C/m² day] 100% of Input Observed Solids Removal 37.3 [g C/m² day] 60.5% of Input Water Column Algal Fixation 1.3 [g C/m² day] 2.08% of Input CO 2 Outgassing 28.8 [g C/m² day] 46.67% of Input Shrimp Output 4.7 [g C/m² day] 7.65% of Input Figure 22: Carbon mass balance; Chemoautotrophic dominated; Photoautotrophic assisted, unit #2, day 116 through day 137. Feed Input 7.0 [g N/m² day] 100% of Input Water Column Algal Fixation 0.2 [g N/m² day] 3.26% of Input Observed Solids Removal 5.7 [g N/m² day] 81.8% of Input Unaccounted N Removal 2.4 [g N/m² day] 34.21% of Input Shrimp Output 1.1 [g N/m² day] 16.01% of Input Figure 23: Carbon mass balance; Chemoautotrophic dominated; Photoautotrophic assisted, unit #2, day 116 through day 137. Page 80

103 Results and Discussion From day 116 to 137 units #1 and #4 the only carbon output was emitted as CO 2 gas. Furthermore, these units discharged no nitrogen other than N 2 resulting from denitrification. One equivalent of alkalinity is generated per equivalent of proteinnitrogen released to produce NH 3 OH. Each equivalent of NH 3 OH N oxidized to nitrate destroys two equivalents of alkalinity, and each equivalent of nitrate reduced to nitrogen gas generates one equivalent of alkalinity. Therefore complete nitrification/denitrification beginning with protein N results in no net change in alkalinity, as shown in equation (35). If no denitrification would take place, an alkalinity destruction of one equivalent of alkalinity per equivalent Nitrogen processed, would have to be substituted as shown in equation (36). (35) with complete Nitrification/denitrification (36) without Nitrification An alkalinity balance was constructed around units #1 and #4 (day 116 to 137). Without denitrification an alkalinity destruction of 0.24 ² for unit #1 and 0.25 ² for Page 81

104 Results and Discussion unit #4, would have been expected. However, unit #1 required only 0.13 unit #4 only ² ² and or 1.55 for unit #1 and 0.76 for unit #4. ² ² To balance the alkalinity destroyed by nitrification and not replaced by bicarbonate addition), 0.12 ² in unit #1 and 0.19 ² in unit #4 or 1.61 and ² 2.63 respectively must have been denitrified to N 2.Since there was no other ² sink for nitrogen, the remaining unaccounted for nitrogen must have accumulated as organic solids within the system. Nitrate and alkalinity balances for unit #1 and #4 (day 116 to 137) are illustrated in Figure 24 and Figure 25. Alkalinity supplement: 1.55(g C/m²/day) or 34.1(g C/m²/22day) or 0.13(eq C/m²/day) or 2.84(eq C/m²/22day) Nitrogen Input: 7.4(g N/m²/day) or 162.8(g N/m²/22day) or 0.53(eq N/m²/day) or 11.6(eq N/m²/22day) Accounted output: 4.0(g N/m²/day) or 87.6(g N/m²/22day) or 0.28(eq N/m²/day) or 6.26(eq N/m²/22day) One m² unit #1 Unaccounted output: 3.4(g N/m²/day) or 75.2(g N/m²/22day) or 0.24(eq N/m²/day) or 5.37(eq N/m²/22day) Alkalinity produced by denitrification: (eq/m²/22day)=2.53(eq/m²/22day) Organic solids accumulation within the system: 2.24(g N/m²/day) or 49.3(g N/m²/22day) or 0.16(eq N/m²/day) or 3.52(eq N/m²/22day) Denitrification: 1.61(g N/m²/day) or 35.4(g N/m²/22day) or 0.12(eq N/m²/day) or 2.53(eq N/m²/22day) Figure 24: Alkalinity mass balance; Chemoautotrophic dominated, unit #1, day 116 through day 137. Page 82

105 Results and Discussion Alkalinity supplement: 0.76(g C/m²/day) or 16.7(g C/m²/22day) or 0.063(eq C/m²/day) or 1.4(eq C/m²/22day) Nitrogen Input: 5.6(g N/m²/day) or 123.2(g N/m²/22day) or 0.4(eq N/m²/day) or 8.8(eq N/m²/22day) Accounted output: 2.0(g N/m²/day) or 45.8(g N/m²/22day) or 0.15(eq N/m²/day) or 3.27(eq N/m²/22day) One m² unit #4 Unaccounted output: 3.5(g N/m²/day) or 77.4(g N/m²/22day) or 0.25(eq N/m²/day) or 5.53(eq N/m²/22day) Alkalinity produced by denitrification: (eq/m²/22day)=4.13(eq/m²/22day) Organic solids accumulation within the system: 0.87(g N/m²/day) or 19.14(g N/m²/22day) or 0.062(eq N/m²/day) or 1.37(eq N/m²/22day) Denitrification: 2.63(g N/m²/day) or 35.4(g N/m²/22day) or 0.19(eq N/m²/day) or 4.13(eq N/m²/22day) Figure 25: Alkalinity mass balance; Chemoautotrophic dominated, unit #4, day 116 through day 137. For the nitrogen mass balance for unit #2 from day 127 to 137, since nitrate concentrations of the reactor outflow were not measured the nitrogen discharge from #3A could not be accounted for. Because of the missing data the amount of nitrogen unaccounted for in this mass balance could not be further specified, it was most likely settled in the denitrification reactor/settling tank or denitrified in the shrimp unit. Negative shrimp outputs resulted from shrimp mortality likely driven by increasing elevated nitrate accumulation of up to 260 during this period. In addition Page 83

106 Results and Discussion sedimentation in the isolated shrimp units #1 and #4 and created isolated anoxic zones, this could have contributed to increased shrimp mortality in these units. Despite higher dissolved oxygen concentrations and shrinking nitrate concentrations, shrimp mortality was high in unit #2 also. Continuously elevated nitrite concentrations of up to 6.8 may have contributed to this. Even though the nitrification reactor was nitrifying during that timeframe, the average difference between in and outflow concentrations of ammonia and nitrite of unit #2 were small. The reason was that the nitrification reactor protected unit #2 from the nitrogen that continued bleeding out of the settling tank into the water column. It was observed that potentially toxic ammonia and nitrite concentrations could be maintained at average concentrations of 1.1 TAN and 0.1 nitrite at feed nitrogen inputs of 7 in the isolated units #1 and #4. However, high solids ² accumulation (up to 980 volatile solids) led to increasing oxygen demand, resulting in lowered dissolved oxygen levels, low as 1.9 (average 3.8 ). Lowest DO concentration in the exchanging unit #2 during that timeframe, with an average of 400 volatile solids, was 3.9 (average 4.7 ). The lack of adequate denitrification in units #1 and #4 led to elevated nitrate concentrations in excess of 300. In addition the isolated shrimp units #1 and #4 Page 84

107 Results and Discussion required continuous alkalinity supplementation of sodium bicarbonate addition of ², or 1.7g of bicarbonate per g of Nitrogen added. In unit #2, exchange with the denitrification reactor/settling tank, prevented excessive nitrate concentrations leading to a decline from end to a final nitrate concentration of 75 ) and no bicarbonate had to be added. Page 85

108 Results and Discussion 4.4 Nitrification Reactor Performance System wide and individual unit or reactor nitrification rates are determined based on either direct measurement of ammonia oxidation (from sub samples) or nitrogen conversion inferred from nitrogen mass balances. Un measurable denitrification could have taken place in anoxic regions of the reactor, consequently, nitrate accumulation could not be used to quantify nitrification rates. Unless otherwise stated, rates are expressed on the area of shrimp production culture footprint (750 m² of PAS) to allow direct comparison feed input rates of the total system. A Total ammonia nitrogen and nitrite balance were developed and presented in Table 38. Table 38: TAN and nitrite balance for the nitrification reactor (days 43-96). Parameter Ave. % of Total inflow (g N/day) TAN inflow Nitrite inflow TAN outflow Nitrite outflow ΔTAN ΔNitrite During this period, the reactor produced no significant reaction of the nitrogen input. At this time the nitrification reactor was divided into two sections, the first aerated and mixed, the second quiescence, to be used as a settling tank. Attempt to return settled Page 86

109 Results and Discussion sludge biomass to the mixed unit repeatedly failed. Because of this lack of the capability to maintain biomass in the mixed zone, the reactor could not significantly impact nitrogen input. Beginning on day 122, the nitrification rector was modified to operate as a sequencing batch reactor, with a fill time of 60 minutes, followed by a mixing time of 20 min, settling time of 20 min and a decant time of 20 minutes. A 2 hp blower was used to mix and aerate the units maintaining biomass in suspension during the fill stage. After settling the accumulated water head (12 cm) was allowed to decant by gravity flow back to the shrimp unit. Distribution of the water to the shrimp units was controlled by elevation of adjustable PVC 90 degree ell fittings placed at the discharge to individual shrimp units #1, #2 and #4. TAN and nitrite balances were developed and are shown in Table 39. Table 39: TAN and nitrite balance for nitrification reactor (days ). Parameter Ave. % of Total inflow (g N/day) TAN inflow Nitrite inflow TAN outflow Nitrite outflow ΔTAN ΔNitrite ΔN Page 87

110 Results and Discussion At this time the nitrification reactor oxidized an average of % of the combined ammonia N and nitrite N inflow to nitrate. This suggests an average ammonia/nitrite oxidation rate of (maximum of ). The reactor was observed to nitrify 1.4 (maximum of 11.1 ) of total shrimp ² ² footprint at a nitrification reactor volatile solids concentration of Within the nitrification reactor ammonia could have been released into the water column from degrading biomass, however this could not be measured, therefore actual ammonia/nitrite oxidation rates are potentially higher than observed. In an attempt to determine total nitrification reactor oxidation rates independent ammonia/ nitrite oxidation rate determinations were conducted on day 137, 147, and 156. The measurements were performed at night to exclude possible photosynthesis processing of nitrogen Table 40: Independently determined nitrification reactor oxidation rates (days 137, 147 and 156). Parameter Ave. (g N/g VS/day) Particulate solids (mg/l) Nitrification at 1000 (mg/l) solids (g N/m²/day) Day Day Day Standard WW treatment plant Page 88

111 Results and Discussion Once reactor particulate solids concentration could be maintained at 1000 the reactor oxidation capacity is projected to average 0.067, 13.5 ², or 66.7(mg N/L reactor day), or ². Therefore an oxidation of 54,621 to nitrate can be achieved through the nitrification reactor operating at 1000 particulate solids concentration. In comparison the total nitrogen Input from feed application to the system averaged 16,188 (36,867 maximum). Page 89

112 Results and Discussion 4.5 Settling Tank/Denitrification Reactor Performance To determine the denitrification capacity of the settling tank/denitrification reactor (unit #3A) total alkalinity and total nitrate nitrogen mass balances were constructed around unit #3. It was assumed that no nitrification took place in the anoxic reactor, therefore nitrate was not produced and alkalinity was not destroyed by nitrification. These mass balances are presented in Table 41. Table 41: Denitrification reactor carbonate mass balance (days ). Parameter Carbonate input r CIC Carbonate inflow r CIFC Ave. (meq/l/d ay) Ave. (g C/m²/day) Min. (g C/m²/day) Max. (g C/m²/day) % of Total input Carbonate outflow r COFC ΔCarbonate r CΔC The corresponding observed decrease in reactor nitrate is shown in the nitrate mass balance illustrated in Table 42. Page 90

113 Results and Discussion Table 42: Denitrification reactor nitrate mass balance (days 129-day 137). Parameter Nitrate input r NIN Nitrate inflow r NIFN Nitrate outflow r NOFN Ave. (mg N/Lreactor/day) Ave. (g N/m²/day) Min. (g N/m²/day) Max. (g N/m²/day) ΔNitrate r NΔN % of Total input A 14 % (25.35 ) increase in alkalinity suggests a rate of denitrification of (per reactor detention time). In average the nitrate concentration across the reactor was reduced by 60% suggesting an average removal rate of 40,469 was achieved through the denitrification reactor, or 30.05(meq) of nitrogen reduced per 25.35(meq) of alkalinity produced. In comparison the total nitrogen Input from feed application to the system averaged 16,188 (36,867 maximum). These measurements and calculations show that in the 2007 PAS system configuration the existing denitrification process provided an average capacity to oxidize an equivalent feed nitrogen application rate of 10 ² (at an average concentration of 47 at 60% efficiency) to nitrogen gas with alkalinity maintenance requiring no alkalinity (NaHCO 3 ) supplementation to the system. Page 91

114 Results and Discussion 10 ² 117 Nitrate 10 ² 47 Nitrate Figure 26: Average nitrate mass balance for unit #3; Mass balance day 129 through day 137 (Denitrifying). Page 92

115 Results and Discussion 4.6 Settling Tank/Denitrification Reactor Sludge Analysis In an attempt to quantify sludge accumulation and total nitrogen content in the settling tank/denitrification reactor (unit #3A), samples at different depths in the tank were obtained on day 151. Volatile solids concentration and TKN content on these samples are shown in Figure 27 and Figure 28. TKN, mg/l Total TKN (mg/l) Depth, cm Figure 27: TKN versus depth in Settling Tank/Denitrification reactor. Volatile solids, mg/l Volatile solids (mg/l) Depth, cm Figure 28: Solids concentration versus depth in Settling Tank/Denitrification reactor. Page 93

116 Results and Discussion A sludge depth profile and water flow path for the settling tank/denitrification reactor was developed (Figure 29). Figure 29: Flow path and sludge elevation in Settling Tank/Denitrification. With an average 20cm sludge layer accumulated on the bottom of the Settling Tank/Denitrification reactor, or 6136 of volatile solids concentration with 1200 TKN concentration, at an area of 166.7m², a total of 40,008(g N) were stored in this sediment. This suggests (based on 750m² culture area and 101 days biomass settling), that at least 0.53 ² was stored. This measurement was obtained post harvest and unmeasured amount of nitrogen had already exited the Page 94

117 Results and Discussion denitrification reactor/settling tank during the culture season. The sludge analysis showed that large amounts of nitrogen were stored in unit #3A in at a shallow depth. This shallow depth proved too easy to get upset as the two events on day 43 and 101 demonstrated. Partial mixing of the sediment or escaping tilapia can and will lead to ammonia discharges resulting in potential failures of the shrimp production system. This reactor design and configuration must be improved to reduce failure of the system by both increasing settling tank/denitrification reactor depth to avoid mixing and locating this reactor to discharge directly to the nitrification reactor as opposed to the shrimp units. Page 95

118 Projected Nitrogen Balance and Proposed System Design 5 PROJECTED NITROGEN BALANCE AND PROPOSED SYSTEM DESIGN 5.1 Projected Nitrogen Balance Based on the previous analysis maximum sustainable nitrogen processing rates are projected for the different system compartments (all rates based on shrimp culture footprint). 1) Denitrification reactor; maximum of 10 concentration of 47 and 2[day] retention time). 2) Nitrification reactor; maximum of 10 3) A shrimp output of 2.8 4) Algal fixation; maximum of algal biomass age. ² ² ² ² (at a Nitrate (at 750 ). (with 2/1(lb feed/lb shrimp)). 2 (at 300 yielding 6 day This suggests a projected average nitrogen balance as shown in Figure 30. Page 96

119 Projected Nitrogen Balance and Proposed System Design Feed Input max 12 [g N/m² day] 100% of Input Water Column Algal Fixation max 2 [g N/m² day] 16.7% of Input Nitrification/ Denitrification Output 9.2 [g N/m² day] 77% of Input Shrimp Output max 2.8 [g N/m² day] 23% of Input Figure 30: Projected average nitrogen balance for optimal combined photoautotrophic/ chemoautotrophic PAS culture of marine shrimp. 5.2 Proposed System Configuration The results show, that the 2007 system configuration was not optimal. Mixing of the settling tank/denitrification reactor led to elevated ammonia concentration on two occasions resulting in high shrimp mortality. The proposed system design offers improvement in system design and configuration to avoid these problems. This work showed that the denitrification reactor was not deep enough and settled solids were easily mixed leading to ammonia loading of the system at large. The recommended of the denitrification/settling is double the actual depth or 1.20m. The depth of the shrimp production (0.6m) depth proved to be adequate. Existing water volumes and microbial Page 97

120 Projected Nitrogen Balance and Proposed System Design biomass levels were capable of processing added feed nitrogen. The proposal system stipulates volumes and areas of the different compartments Table 43. Table 43: Proposed 1 ha shrimp production system component areas and volumes. Compartment Depth Volume % total of Volume Area % of total area [m] [m³] [%] [m²] Shrimp units Nitrification units Denitrification units Tilapia units [%] To isolate potential ammonia discharges from the settling tank/denitrification reactor, this reactor is proposed to be located as a separate exchange loop tied to the nitrification reactor. Water from the tilapia zone will discharge directly into the setting tank with water from the settling tank/denitrification reactor discharging to the nitrification reactor. However, in the event of an upset or overloading the denitrification reactor can be bypassed. In Figure 31 the proposed system configuration is shown, were 1) is Denitrification/Settling reactor, 2) is Nitrification reactor, 3) is Tilapia confined unit, and 4) are Shrimp production units, the arrows show the flow direction. Page 98

121 Projected Nitrogen Balance and Proposed System Design Feed input Commercial feed; 32% Protein Max: 2343 (kg/ha/day) Shrimp Units (4) 60% of volume 73.8% of area Stocking: 1200 (Shrimp/m²) Survival: 70% Ѳ= 1 (exchange/day) Photoautotrophic =300 (mg/l) Flow Q=4428 (m³/ha day) Tilapia Unit (3) 6.6% of volume 8.3% of area Stocking: 25% of Shrimp final density Ѳ= 9 (exchange/day) Photoautotrophic Flow Q=4428 (m³/ha day) Flow Q=4428 (m³/ha day) Nitrification Reactor (SBR) (2) Ѳ= 3 (exchange/day) Chemoautotrophic =750 (mg/l) Denitrification Unit (1) Ѳ= 4.5 (exchange/day) Heterotrophic, anoxic =~20cm (sediment) Flow Q=4428 (m³/ha day) Figure 31: Proposed optimal PAS photoautotrophic/ chemoautotrophic system design. To lower the risk of impact resulting from mixing of the denitrification reactor and to optimize the flow patterns for component substances (alkalinity, nitrate) the denitrification reactor is proposed to exchange with the nitrification reactor in a separate loop. Shrimp units will discharge to tilapia unit to insure VS density and algal species control. Discharge from the tilapia unit flows to either denitrification reactor or Page 99

122 Projected Nitrogen Balance and Proposed System Design nitrification reactor for ammonia and nitrite control and then from nitrification reactor back to shrimp units. The proposed exchange rates are: One exchange per day in the shrimp units, 9 exchanges per day in the Tilapia units, 3 exchanges per day in the nitrification rectors, and 4.5 exchanges per day in the denitrification reactors. Maximum feed assimilation capacity is projected at 2,343 (2,109 ). Average seasonal feed loading is 50% of maximum yielding a seasonal maximum projected feed application of 1,171.9 (1,054.5 ), or 175,785 (158,175 ) of feed application for a 150 days growing season. Projected shrimp yields at a 2/1 conversion are 87,889 or 79, days growing season. This corresponds to ² of 15(gm) harvest size animals. ² for a or Actual shrimp biomass yields obtained from previous years are compared with the projected optimal system (Table 44). Table 44: Past and proposed shrimp yield, season length and feed rates. Year Shrimp Biomass Season Feed Yield Maximum Capacity length Average Peak (lb/ac) (lb/ac) (Day) (lb/ac/day) (lb/ac/day) , , , , proposed 79, , Page 100

123 Summary and Conclusions 6 SUMMARY AND CONCLUSIONS 6.1 Summary The objective of this research was to further modify and develop the Clemson Partitioned Aquaculture System (PAS) targeting the economic production of marine shrimp, (Litopenaeus vannamei), production in excess of 45,000 (40,000 ) within a 5 month culture period while minimizing the impact on the surrounding environment. The PAS included three 250m² (151m³) shrimp units, one 83m² (50m³) unit stocked with Nile tilapia (Oreochromis niloticus) for control of algal species composition and density, one 167m² (100m³) denitrification/settling tank, and later in the season, a single 49m² (151m³) high rate nitrification reactor was added to the system flow path. The units were enclosed within a covered sunlight permeable greenhouse with mechanically adjustable sides and thermostatically operated fan for temperature control. Six blade paddlewheels were used to impart a uniform water velocity of 0.12 (0.4 ) directed by plastic curtains fixed along the central length yielding a water circulation in an ovular circuit. One third HP sump pumps were used to transfer water between units. Water levels were maintained at 60cm by using float switches to sense water levels within each unit. The operation of the high rate pilot scale shrimp culture system included stocking of shrimp post larvae (PL 8/9), feeding three times per day at the maximum possible rate Page 101

124 Summary and Conclusions restricted by water quality and shrimp uptake, measuring water quality three times per day, adjustments of system flow path as needed or as dictated by experimental design, ph control with CO 2 addition, and/or carbonate addition when required, and final harvest of shrimp biomass. The units were stocked with 1000 on June 13, Because of high initial ² mortalities units were restocked at day 12 with an additional 400. Final overall harvest density averaged 207. Accumulation of excessive solids and resultant ² high ammonia concentrations in the settling tank/denitrification reactor led to high ammonia and nitrite concentrations in the shrimp units, leading to major shrimp mortality on day 34 and 101. Late season high nitrate levels further increased mortality in the shrimp units. An overall average shrimp biomass of 23,400 (20,800 ) was harvested at the end of the season (149 days post stocking) yielding average individual shrimp harvest weights of (649 ). The overall average feed rate applied to the system was ), with peak feed rates of (1645 ² ). In the first third of the season, water was pumped from the shrimp units through the settling tank and tilapia unit. On day 34 high ammonia levels were produced in the settling tank as a result of excessive mixing. Following this incident, the exchange of the shrimp units was suspended for 5 days as ammonia and nitrite levels exceeded 5. Page 102

125 Summary and Conclusions Also at this time a separate independent nitrification reactor operated as aerated/settling combination was brought on line and connected to the system. Beginning on day 43 all shrimp units were exchanged with the denitrification reactor/settling tank, tilapia confined unit, and finally nitrification reactor. The microbial solids content of the system was dominated by algal biomass from day 1 to day 101, until the exchange was suspended on 101. At that time ammonia concentrations in the settling tank again climbed to in excess of 8. After two weeks of isolated operation the shrimp units microbiological composition changed from algal to chemoautotrophic domination with 500 of non algal bioflocs suspension in the water column. On day 116 the nitrification reactor was reconfigured to operate as a sequencing batch reactor, with a 60 min fill, 20 min reaction, 20 min settle, and 20 min decant stage. At this time water circulation was reestablished between shrimp unit #2, the settling tank/denitrification reactor, tilapia tank, and nitrification reactor. In contrast, units #1 and #4 continued to operate as isolated grow outs units for the remainder of the experiment. Alkalinity production from denitrification eliminated the need for system alkalinity addition during periods of continuous recirculation. When isolated, the individual units required or 1.7g of bicarbonate addition per gram of nitrogen addition to the system. The data from system operation collected throughout the season was reduced and analyzed. Carbon and nitrogen mass balances for selected representative periods Page 103

126 Summary and Conclusions representing steady state performance were constructed to provide a quantitative understanding of interactions of algal and bacterial communities within the system. During the algal dominated period of day 43 through day 96 the hydraulic detention time ranged from 0.5/day in unit #1, 1.3/day in unit #2 to 1.0/day in unit #4 and had insignificant influence on water quality, solids concentration or photosynthesis (VS of 277 ±20, photosynthesis of 59.4( ). Feed applications averaged 99.6% (39.12 ) with recarbonation additions via ² CO 2 injection accounting for 0.4% (0.16 ) of carbon input to the system. 34.1% ² of added carbon (13.38 ) was fixed by algae in the water column. Shrimp ² biomass accounted for 9% (3.45 ) while observed solids removal accounted for ² 16.9% (6.64 ² of carbon removal. 74% (29.1 ) of carbon was ² unaccounted for, mainly leaving the system as CO 2 or unmeasured sedimentation in the settling tank/denitrification reactor. The single input of nitrogen to the system was from feed application of (4.45 ); 53.7% of this application (2.39 ) was ² ² fixed by algae. Observed solids removal accounted for 25.3% (1.13 ), shrimp ² output for 19% (0.85 ) and 55.8% (2.48 ) was unaccounted for. ² ² Since no nitrogen output other than flow through the denitrification reactor/settling Page 104

127 Summary and Conclusions tank could be account for the remainder of the nitrogen, it was assumed that the unaccounted nitrogen was stored in this reactor. An ammonia and nitrite balance was performed around the nitrification reactor. The nitrifying reactor was observed to yield a capacity to nitrify 13.5 at an average nitrifying biomass concentration of ² A nitrate balance on the denitrification reactor suggested a capacity to remove 10 ², or 36.1 ², or 30.05(meq) of nitrogen reduced per 25.35(meq) of alkalinity produced at nitrate concentrations averaging 47. Based on the proto type performance and operation, a design for an optimal combined photoautotrophic/ chemoautotrophic system for high rate, zero discharge culture of marine shrimp was proposed. The optimal system is projected to process a maximum nitrogen input of 12 that suggesting a maximum feed application rate of 2,343 87,889 ² (2,109 ), yielding a projected shrimp yield of (79,087 season, and this corresponds to (gm) harvest size animals. ² ) within a 150 day growing or 586 ² of Page 105

128 Summary and Conclusions 6.2 Conclusions Shrimp Culture System Prototype Operation and Performance A) Feeding and Production 1) After 149 days of culture, 20,800(lb shrimp biomass/ac of shrimp footprint), and 14900(lb shrimp biomass/ total system footprint), was harvested. 2) Final shrimp weights after 149 days growth with stocking of PL 8/9 averaged 11.3(g/shrimp) overall with 11.0(g/shrimp) in unit #1, 12.4(g/shrimp) in unit #2 and 10.6(g/shrimp) in unit #4. 3) Overall average daily feed rate was 649 (lb/acre/day), ranging from 678 (lb/acre/day) in unit #1, 679(lb/acre/day), in unit #2, and 592(lb/acre/day) in unit #4. 4) Overall maximum daily feed rate was 1645(lb/acre/day), ranging from 1621(lb/acre/day) in unit #1, 1692(lb/acre day) in unit #2, and 1645(lb/acre/day) in unit #4. B) Water Quality and System Mass Balances 5) Three major mortality events, one at stocking, and two related to handling of settled solids resulted in animal densities ranging from 1400 animals/m² at stocking to 754 animals/m² on day 35 to 544 animals/m² on day 102 to an overall average of 207 animals/m² at final harvest. 6) System performance was dominated by algal photosynthesis (processing 53.7% of nitrogen added) from day 43 to 96 and bacterial nitrification (processing 55% of added nitrogen) from day 116 to day ) Net photosynthesis for the algal dominated period ranged from day 1 to day 101 with an overall average of 62.5(mg O 2 /L/day) with 64.5(mg O 2 /L/day) in unit #1, 67.2(mg O 2 /L/day) in unit #2 and 55.7(mg O 2 /L/day) in unit #4. Page 106

129 Summary and Conclusions 8) Peak net photosynthesis for the algal dominated period was 106.9(mg O 2 /L/day), ranging from 105.2(mg O 2 /L/day) in unit #1, 117.4(mg O 2 /L/day) in unit #2 and 98.2(mg O 2 /L/day) in unit #4. 9) Net photosynthesis for the bacterial nitrifying dominated period from day 116 to day 146 with an overall average of 5.4(mg O 2 /L/day), ranging from 8.9(mg O 2 /L/day) in unit #1, 3.7(mg O 2 /L/day) in unit #2 and 11.0(mg O 2 /L/day) in unit #4. 10) Different hydraulic detention times of 0.5/day in unit #1, 1.3/day in unit #2 or 1.0/day in unit #4, had insignificant influence on water quality, solids concentration, and photosynthesis. 11) Overall carbon flux during algal dominated period averaged 39.12(g C/m²/day) feed application (99.6%), 0.16(g C/m²/day) (0.4%) CO2 recarbonation, algal internal fixation of 13.38(g C/m²/day) (34.1%), measured solids discharge of 6.64(g C/m²/day) (16.9%), shrimp harvest of 3.45(g C/m²/day) (9%), and 29.1(g C/m²/day) (74.1%) resulting from CO 2 outgassing and unaccounted for algal and bacterial solids removal. 12) Overall nitrogen flux during algal dominated period averaged 4.45(g N/m²/day) feed application (100%), algal internal fixation of 2.39(g N/m²/day) (53.7%), measured solids discharge of 1.13(g N/m²/day) (25.3%), shrimp harvest of 0.85(g N/m²/day) (19%), and 2.48(g N/m²/day) (55.8%) resulting from unaccounted for nitrification/denitrification and algal and bacterial solids removal. 13) Unit #1 carbon flux during bacterial dominated period averaged 65.1(g C/m²/day) feed application (96.54%), 2.3(g C/m²/day) (3.46%) NaHCO 3 supplement, algal internal fixation of 0(g C/m²/day) (0%), measured solids accumulation of 6.83(g C/m²/day) (10.13%), shrimp harvest of 1.4(g C/m²/day) ( 2.06%), and 61.2(g C/m²/day) (90.83%) resulting from CO 2 outgassing. Page 107

130 Summary and Conclusions 14) Unit #1 nitrogen flux during bacterial dominated period averaged 7.4(g N/m²/day) feed application (100%), algal internal fixation of 0(g N/m²/day) (0%), measured solids accumulation of 1.05(g N/m²/day) (14.19%), measured nitrate accumulation of 3.23(g N/m²/day) (43.59%), shrimp harvest of 0.3(g N/m²/day) ( 4.47%), 1.6(g N/m²/day) (21.6%) denitrification, and 2.24(g N/m²/day) (30.1%) resulting from unaccounted for solids settled within the system. 15) Unit #4 carbon flux during bacterial dominated period averaged 48.9(g C/m²/day) feed application (97.68%), 1.2(g C/m²/day) (2.32%) NaHCO 3 supplement, algal internal fixation of 0(g C/m²/day) (0%), measured solids accumulation of 3.56(g C/m²/day) (7.11%), shrimp harvest of 1.9(g C/m²/day) ( 3.80%), and 50.8(g C/m²/day) (101.48%) resulting from CO 2 outgassing. 16) Unit #4 nitrogen flux during bacterial dominated period averaged 5.6(g N/m²/day) feed application (100%), algal internal fixation of 0(g N/m²/day) (0%), measured solids accumulation of 0.55(g N/m²/day) (9.84%), measured nitrate accumulation of 1.93(g N/m²/day) (34.72%), shrimp harvest of 0.5(g N/m²/day) ( 8.14%), 2.6(g N/m²/day) (46.5%) denitrification, and 0.87(g N/m²/day) (15.5%) resulting from unaccounted for solids settled within the system. 17) Unit #2 carbon flux during bacterial dominated period averaged 61.7(g C/m²/day) feed application (100%), 0(g C/m²/day) (0%) NaHCO 3 supplement, algal internal fixation of 1.3(g C/m²/day) (2.08%), measured solids removal of 37.3(g C/m²/day) (60.5%), shrimp harvest of 4.7(g C/m²/day) ( 7.65%), and 28.8(g C/m²/day) (46.67%) resulting from CO 2 outgassing. 18) Unit #2 nitrogen flux during bacterial dominated period averaged 7.0(g N/m²/day) feed application (100%), algal internal fixation of 0.2(g N/m²/day) (3.26%), measured solids discharge of 5.7(g N/m²/day) (81.8%), shrimp harvest of 1.1(g N/m²/day) ( 16.01%), and 2.4(g N/m²/day) (34.21%) resulting from unaccounted for nitrification/denitrification. Page 108

131 Summary and Conclusions C) Settling Tank/Denitrification Reactor, and Nitrifiying SBR Performance 19) The settling tank/denitrification reactor was under designed both in surface area and depth (166.7 m² and 0.6m). Total solids concentration (up to 25cm from bottom) in settling tank reached levels of 8000 (mg/l) (unit #3A). Unplanned mixing of the settling tank led to major mortality events bleeding 5 and 9 (mg/l) of ammonia concentration to system at large on days 34 and ) On day 151, 40,008(g N) were still stored in denitrification reactor/settling tank, or 240(g/m²), or 53.5(g N/m² shrimp footprint), or 0.53(g N/m² shrimp footprint/day) 21) Sequencing batch nitrification reactor was successfully operated with a fill cycle time of 60 minutes, followed by 20 minutes of reacting, 20 minutes of settling, and finally 20 minutes of decant operating at a solids concentration of 500 to 900(mg/l) 22) SBR nitrogen processing rate averaged 0.067(g N/g VS/day), or 67(mg N/l/day) or 13.5(g N/m² shrimp footprint/day) at 1000 mg/l volatile solids concentration and 2 days detention time. 23) Denitrification reactor averaged 60%, nitrogen removal at a rate of 36.1(g N/m²reactor/day), or 60.1(mg N/l/day), or 10(g N/m² shrimp footprint/day), or 30.05(meq) of nitrogen reduced per 25.35(meq) of alkalinity produced. 24) Alkalinity production from denitrification eliminated the need for system alkalinity addition during periods of continuous exchange. When isolated the individual units required or 1.7g of bicarbonate addition per gram of nitrogen addition to the system Page 109

132 Summary and Conclusions Proposed Shrimp System Configuration, Capacity, Yield, and Operation 25) Proposed photoautotrophic/ chemoautotrophic system for high rate, zerodischarge culture of marine shrimp suggests 73.8% of total system area devoted to shrimp 9.8% for nitrification SBR, 8.2% for settling tank/denitrification units, and 8.2% for tilapia confinement. 26) Recommended system design depths are 1.2m for settling tank/denitrification reactor, 0.6m for shrimp culture units, 0.6m for tilapia confinement zone and, 1.5m for the nitrification reactor. 27) Shrimp units recommended configuration discharges directly to tilapia zone, followed by discharge to denitrification reactor, followed by discharge to nitrification reactor returning to shrimp units. 28) Recommended exchange rates are once/day in shrimp units, 9/day in tilapia zone, 3 /day in nitrification units, and 4.5/day in denitrification reactor. 29) Projected nitrogen mass balance for proposed system is maximum nitrogen input of 12(g N/m²/day) feed application, algal fixation of 2(g N/m²/day) (16.7% of input), recovery of 2.8(g N/m²/day) (23% of input) as shrimp biomass, and discharge/treatment of 9.4 (g N/m²/day) (77% of input) through nitrification/denitrification. 30) Projected system maximum feed processing capacity is 2,343(kg/ha/day) or 2,109(lb/ac/day), corresponding to (12 g N/m2/day), resulting in a seasonal average of 1,054.5 (lb/ac/day), or 6 (g N/m²/day). 31) The maximum carrying capacity of the proposed system is 175,775kg/ha 150day (158,174(lb/ac 150day), corresponding to a seasonal net yield of 87,889 kg/ha 150day (79,087(lb/ac 150day), 8.8 (kg shrimp/m2) or 586 (shrimp/m2) of 15(gm). Page 110

133 APPENDICES

134 Appendices A. Abbreviations Table 45: Abbreviations. Abbreviation PAS SBR N C Anammox ANX AER gpm DO TSS VS TNVS TKN TAN DI ABS Trans Hi Lo SAL SD ALK C:N inf. WW Meaning Partitioned Aquaculture System Sequence Batch Reactor Nitrogen Carbon Anaerobic Ammonia Oxidation Anoxic Aerobic Gallon per minute Dissolved Oxygen Total Suspendable Solids Volatile Solids Total Non Volatile Solids Total Kjeldahl Nitrogen Total Ammonia Nitrogen De Ionized Absorption Transmission High Level Low Level Salinity Secchi depth Alkalinity Carbon Nitrogen ratio Infinite Waste water Page 112

135 Appendix B. Symbols Table 46: Symbols. Identifier Name Unit Q Flow rate Δt Time Interval ρ Density c Concentration x Concentration r Rate per area V Volume T Temperature A Area ² Δfeed Total amount during timeframe VS Volatile Solids Table 47: Indices. Indices Meaning w Water 0 Initial f Final in Inflow out Outflow t At time t 1 At time 1 2 At time 2 3 At time 3 CIF Carbon Input by Feed application CIR Carbon Input by Recarbonation CIC Carbon Input by Carbonate Input CIS Carbon Input by Sugar Input CSA Carbon Storage by Algal Fixation Page 113

136 Appendix CSS CSC COS COC CIFC COFC CΔC ww NIF NSA NSS NSN NON NOS NIFN NOFN NΔN Carbon Storage by Sludge Accumulation Carbon Storage by Carbonate Accumulation Carbon Output by Shrimp Carbon Output by CO 2 Outgassing Carbonate Carbon Inflow Carbonate Carbon Outflow Difference in Carbonate Flows Wet weight Nitrogen Input by Feed Application Nitrogen Storage by Algal Fixation Nitrogen Storage by Sludge Accumulation Nitrogen Storage due to Nitrate Accumulation Nitrogen Output by Nitrification/Denitrification or Unaccounted Nitrogen Output by Shrimp Nitrate Nitrogen Inflow Nitrate Nitrogen Outflow Difference in Nitrate Flows Page 114

137 Appendix C. Unit Conversions Table 48: Metric to American unit conversions. Metric American 1 cm = in 1 m = 3.28 ft 1 kg = lb 1 ha = 2.5 ac 1 L = gal 1 W = US Hp Page 115

138 Appendix D to 2005 PAS Configuration and Water Circulation Figure 32 to Figure 34 illustrate the changing PAS configurations for the years 2003, 2004 and The PAS configuration of the early season in 2007 is presented in Figure 12 and the final PAS configuration for the year 2007 is shown in Figure 13. Figure 32: 2003 PAS configuration. Source: Redrwan from Brune, Page 116

139 Appendix Figure 33: 2004 PAS configuration. Source: Redrawn from Brune, Figure 34: 2005 PAS configuration. Source:Redrawn from Brune, Page 117

140 Appendix In 2003 the PAS system contained three shrimp grow out units and one tilapia confined unit. The water from the shrimp units was exchanged with the tilapia unit for algal biomass removal. In this system nitrogen was controlled by primarily algal growth and biomass removal. In 2004 anoxic zones were added to unit #1 and #4 to study the impact of adding higher density nitrifying reactors to the system. Unit #2 was maintained as a control without anoxic zone. In this system nitrogen was controlled by a combination of algal growth and nitrification (Kirk, 2004). In 2005 the system configuration was modified by adding an anaerobic and aerated zone. The aerated zone is named #3A and the anaerobic zone is named #3C. This system combined the nitrification/ denitrification process with reduced algal production to process nitrogen. Page 118

141 Appendix E. Obtained Data Shrimp Unit #1 Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 35: Event timeline for unit # Page 119

142 Appendix Food Input as Forcing Function: Feed input (lb/ac/day) feed rate (lb/ac/day) Feed Input 7 day average (lb/ac/day) 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 36: Food input rate for unit # Feed rate (1000 g/day) Macro Visualization: Micro Visualization: Page 120

143 Appendix Water Circulation From reactor From unit #3 To unit #3 To unit #3C Days from stocking Figure 37: Water circulation chart from unit #1. Water Exchanges Per Day Exchanges per day /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 38: Water exchanges per day unit #1. Exchanges per day 2 day average Page 121

144 Appendix Temperature (T) Temp (oc) 7 day Temp (oc) Temp (oc) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 39: Daily and 7-day average temperature in unit #1. Dissolved Oxygen (DO) DO (mg/l) 7 day DO (mg/l) DO (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 40: Daily and 7-day average dissolved oxygen concentration in unit # Page 122

145 Appendix Salinity (Sal) Salinity (g/l) Salinity (g/l) 7 day Salinity (g/l) 2 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 41: Daily and 7-day average salinity in unit #1. PH ph ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 42: Daily and 7-day average ph in unit #1. ph 7 day ph ( ) CO CO2 Time (min) Page 123

146 Appendix Secchi Depth (SD) Secchi (cm) 7 day Secchi Secchi Depth (cm) /13/07 7/4/07 7/25/07 8/15/07 Date/Time 9/5/07 9/26/07 10/17/07 11/7/07 Figure 43: Daily and 7-day average secchi depth in unit #1. Volatile Solids (VS) Volatile solids (mg/l) Solids (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 44: Weekly Solids in unit # Page 124

147 Appendix Alkalinity (ALK) Alkalinity (mg/l) Alkalinity (mg/l) NaHCO3 intake (lb) NaHC03 intake (lb) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 45: Alkalinity and NaHCO 3 supplement in unit #1. Total Ammonia Nitrogen (TAN) TAN (mg N/L) TAN (mg N/L) 7 day TAN (mg N/L) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 46: Daily and 7-day average total ammonia nitrogen in unit # Page 125

148 Appendix Unionized Ammonia (NH 3 N) Unionized Ammonia (mg N/L) Unionized Ammonia (mg N/L) 7 day Unionized Ammonia (mg N/L) 0.0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 47: Daily and 7-day average unionized ammonia in unit #1. Nitrite (NO 2 N) NO2 (mg N/L) 7 day NO2 (mg N/L) NO2 (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 48: Daily and 7-day average nitrite in unit # Page 126

149 Appendix Nitrate (NO 3 N) NO2 (mg N/L) NO3 (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 49: Daily and 7-day average nitrate in unit #1. Total Kjeldahl Nitrogen (TKN) Total TKN (mg/l) Soluble TKN (mg/l) TKN (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 50: Total Kjeldahl and particulate organic nitrogen in unit # Page 127

150 Appendix Particulate Carbon Nitrogen ratio (C:N ratio) Calculated C:N ratio C:N ratio ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 51: Particulate carbon-nitrogen ratios(c:n-ratio) in unit #1. Net Algal Photosynthesis and Water Column Respiration O 2 (mg/l/day) Net algal photosynthesis Water column respiration 40 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 52: Net algal photosynthesis and water column respiration in unit # Page 128

151 Appendix Shrimp Density Projected from Food Consumption Figure 53: Shrimp density projected from food consumption and measured shrimp size in unit #1. Figure 54: Projected shrimp biomass in unit # Page 129

152 Appendix F. Obtained Data Shrimp Unit #2 Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 55: Event timeline for unit # Page 130

153 Appendix Food Input as Forcing Function: Feed input (lb/ac/day) feed rate (lb/ac/day) Feed Input 7 day average (lb/ac/day) 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 56: Food input rate for unit # Feed rate (1000 g/day) Macro Visualization: Micro Visualization: Page 131

154 Appendix Water Circulation From reactor From unit #3 To unit #3 To unit #3A To unit #3C Days from stocking Figure 57: Water circulation chart from unit #2. Water Exchanges Per Day Exchanges per day Exchanges per day 2 day average /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 58: Water exchanges per day in unit # Page 132

155 Appendix Temperature (T) Temp (oc) 7 day Temp (oc) Temp (oc) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 59: Daily and 7-day average temperature in unit #2. Dissolved Oxygen (DO) DO (mg/l) DO (mg/l) 7 day DO (mg/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 60: Daily and 7-day average dissolved oxygen concentration in unit # Page 133

156 Appendix Salinity (Sal) Salinity (g/l) Salinity 7 day Salinity (g/l) 2 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 61: Daily and 7-day average salinity in unit #2. PH ph ( ) /13 7/4 7/25 8/15 Date/Time 9/5 9/26 10/17 11/7 Figure 62: Daily and 7-day average ph in unit #2. ph 7 day ph ( ) CO CO2 Time (min) Page 134

157 Appendix Secchi Depth (SD) Secchi (cm) 7 day Secchi (cm) Secchi Depth (cm) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 63: Daily and 7-day average secchi depth in unit #2. Volatile Solids (VS) Volatile solids (mg/l) Solids (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 64: Weekly Solids in unit # Page 135

158 Appendix Alkalinity (ALK) Alkalinity (mg/l) Alkalinity (mg/l) NaHCO3 intake (lb) NaHC03 intake (lb) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 65: Alkalinity and NaHCO 3 supplement in unit #2. Total Ammonia Nitrogen (TAN) TAN (mg N/L) 7 day TAN (mg N/L) TAN (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 66: Daily and 7-day average total ammonia nitrogen in unit # Page 136

159 Appendix Unionized Ammonia (NH 3 N) Unionized Ammonia (mg N/L) Unionized Ammonia (mg N/L) 7 day Unionized Ammonia (mg N/L) 0.0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 67: Daily and 7-day average unionized ammonia in unit #2. Nitrite (NO 2 N) NO2 (mg N/L) 7 day NO2 (mg N/L) NO2 (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 68: Daily and 7-day average nitrite in unit # Page 137

160 Appendix Nitrate (NO 3 N) NO2 (mg N/L) NO3 (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 69: Daily and 7-day average nitrate in unit #2. Total Kjeldahl Nitrogen (TKN) and Particulate Organic Nitrogen TKN (mg/l) Total TKN (mg/l) Soluble TKN (mg/l) Particulate organic N (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 70: Total Kjeldahl and particulate organic nitrogen unit # Page 138

161 Appendix Particulate Carbon Nitrogen ratio (C:N ratio) Calculated C:N ratio C:N ratio ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 71: Particulate carbon-nitrogen ratios (C:N-ratio) in unit #2. Net Algal Photosynthesis and Water Column Respiration O 2 (mg/l/day) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/ Time Figure 72: Net algal photosynthesis and water column respiration in unit #2. Net algal photosynthesis Water column respiration Page 139

162 Appendix Shrimp Density Projected from Food Consumption Figure 73: Shrimp density projected from food consumption and measured shrimp size in unit #2. Figure 74: Projected shrimp biomass in unit # Page 140

163 Appendix G. Obtained Data Tilapia confined Unit #3 Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 75: Event timeline for unit # Page 141

164 Appendix Macro Visualization: Micro Visualization: Page 142

165 Appendix Water Circulation From unit #3A From/to unit #4 From/to unit #2 From/to unit #4 To reactor Days from stocking Figure 76: Water circulation chart from unit #3. Water Exchanges Per Day Exchanges per day /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 77: Water exchanges per day in unit #3. Exchanges per day 2 day average Page 143

166 Appendix Temperature (T) Temp (oc) 7 day Temp (oc) Temp (oc) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 78: Daily and 7-day average temperature in unit #3. Dissolved Oxygen (DO) DO (mg/l) DO (mg/l) 7 day DO (mg/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 79: Daily and 7-day average dissolved oxygen concentration in unit # Page 144

167 Appendix Salinity (Sal) Salinity (g/l) Salinity 7 day Salinity (g/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 80: Daily and 7-day average salinity in unit #3. PH ph ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 81: Daily and 7-day average ph in unit #3. ph 7 day ph ( ) CO CO2 Time (min) Page 145

168 Appendix Secchi Depth (SD) Secchi (cm) 7 day Secchi (cm) Secchi Depth (cm) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 82: Daily and 7-day average secchi depth in unit #3. Volatile Solids (VS) Volatile solids (mg/l) Solids (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 83: Weekly Solids in unit # Page 146

169 Appendix Alkalinity (ALK) Alkalinity (mg/l) Alkalinity (mg/l) NaHCO3 intake (lb) NaHC03 intake (lb) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 84: Alkalinity and NaHCO 3 supplement in unit #3. Total Ammonia Nitrogen (TAN) TAN (mg N/L) 7 day TAN (mg N/L) TAN (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 85: Daily and 7-day average total ammonia nitrogen in unit # Page 147

170 Appendix Unionized Ammonia (NH 3 N) Unionized Ammonia (mg N/L) Unionized Ammonia (mg N/L) 7 day Unionized Ammonia (mg N/L) 0.0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 86: Daily and 7-day average unionized ammonia in unit #3. Nitrite (NO 2 N) NO2 (mg N/L) NO2 (mg N/L) 7 day NO2 (mg N/L) 2 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 87: Daily and 7-day average nitrite in unit # Page 148

171 Appendix Total Kjeldahl Nitrogen (TKN) TKN (mg/l) Total TKN (mg/l) Soluble TKN (mg/l) Particulate organic N (mg/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 88: Total Kjeldahl and particulate organic nitrogen unit #3. Particulate Carbon Nitrogen ratio (C:N ratio) Calculated C:N ratio C:N ratio ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 89: Particulate carbon-nitrogen ratios in unit # Page 149

172 Appendix Net Algal Photosynthesis and Water Column Respiration O 2 (mg/l/day) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/ Time Figure 90: Net algal photosynthesis and water column respiration in unit #3. Net algal photosynthesis Water column respiration Page 150

173 Appendix H. Obtained Data Denitrification Reactor/Settling Tank (Unit #3A) Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 91: Event timeline for unit #3A Page 151

174 Appendix Total Ammonia Nitrogen (TAN) TAN (mg N/L) TAN (mg N/L) 7 day TAN (mg N/L) 5 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 92: Daily and 7-day average total ammonia nitrogen in unit #3A. Nitrite (NO 2 N) NO2 (mg N/L) NO2 (mg N/L) 7 day NO2 (mg N/L) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 93: Daily and 7-day average nitrite in unit #3A Page 152

175 Appendix I. Obtained Data Shrimp Unit #4 Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 94: Event timeline for unit # Page

176 Appendix Food Input as Forcing Function: Feed input (lb/ac/day) feed rate (lb/ac/day) Feed Input 7 day average (lb/ac/day) 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 95: Food input rate for unit # Feed rate (1000 g/day) Macro Visualization: Micro Visualization: Page

177 Appendix Water Circulation From reactor From unit #3 To unit #3 To unit #3C Days from stocking Figure 96: Water circulation chart from unit #4. Water Exchanges Per Day Exchanges per day Figure 97: Water exchanges per day in unit #4. Exchanges per day 2 day average 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Page

178 Appendix Temperature (T) Temp (oc) 7 day Temp (oc) Temp (oc) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 98: Daily and 7-day average temperature in unit #4. Dissolved Oxygen (DO) DO (mg/l) DO (mg/l) 7 day DO (mg/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 99: Daily and 7-day average dissolved oxygen concentration in unit # Page

179 Appendix Salinity (Sal) Salinity (g/l) Salinity 7 day Salinity (g/l) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 100: Daily and 7-day average salinity in unit #4. PH ph ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 101: Daily and 7-day average ph in unit #4. ph 7 day ph CO CO2 Time (min) Page

180 Appendix Secchi Depth (SD) Secchi (cm) 7 day Secchi (cm) Secchi Depth (cm) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 102: Daily and 7-day average secchi depth in unit #4. Volatile Solids (VS) Volatile solids (mg/l) Solids (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 103: Weekly Solids in unit # Page

181 Appendix Alkalinity (ALK) Alkalinity (mg/l) Alkalinity (mg/l) NaHCO3 intake (lb) NaHC03 intake (lb) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 104: Alkalinity and NaHCO 3 supplement in unit #4. Total Ammonia Nitrogen (TAN) TAN (mg N/L) 7 day TAN (mg N/L) TAN (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 105: Daily and 7-day average total ammonia nitrogen in unit # Page

182 Appendix Unionized Ammonia (NH 3 N) Unionized Ammonia (mg N/L) Unionized Ammonia (mg N/L) 7 day Unionized Ammonia (mg N/L) 0.0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 106: Daily and 7-day average unionized ammonia in unit #4. Nitrite (NO 2 N) NO2 (mg N/L) NO2 (mg N/L) 7 day NO2 (mg N/L) 0 6/13/07 7/4/07 7/25/07 8/15/07 9/5/07 Date/Time 9/26/07 10/17/07 11/7/07 Figure 107: Daily and 7-day average nitrite in unit # Page

183 Appendix Nitrate (NO 3 N) NO3 (mg N/L) NO3 (mg N/L) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 108: Daily and 7-day average nitrate in unit #4. Total Kjeldahl Nitrogen (TKN) TKN (mg/l) Total TKN (mg/l) Soluble TKN (mg/l) Particulate organic N (mg/l) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 109: Total Kjeldahl and particulate organic nitrogen unit # Page

184 Appendix Particulate Carbon Nitrogen ratio (C:N ratio) Calculated C:N ratio C:N ratio ( ) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/Time Figure 110: Particulate carbon-nitrogen ratios (C:N-ratio) of unit #4. Net Algal Photosynthesis and Water Column Respiration O 2 (mg/l/day) /13/07 7/4/07 7/25/07 8/15/07 9/5/07 9/26/07 10/17/07 11/7/07 Date/ Time Figure 111: Net algal photosynthesis and water column respiration in unit #4. Net algal photosynthesis Water column respiration Page

185 Appendix Shrimp Density Projected from Food Consumption Figure 112: Shrimp density projected from food consumption and measured shrimp size in unit #4. Figure 113: Projected shrimp biomass in unit # Page

186 Appendix J. Obtained Data Nitrification Reactor Operational Timeline Days from stocking: Months of season: November 07 October 07 September 07 August 07 July 07 June 07 Figure 114: Event timeline for the reactor Page 164

187 Appendix Macro Visualization: Micro Visualization: Page 165