Understanding photoautotrophic, autotrophic, and heterotrophic bacterial based systems using basic water quality parameters

Transcription

1 Understanding photoautotrophic, autotrophic, and heterotrophic bacterial based systems using basic water quality parameters Abstract James M. Ebeling Aquacultural Engineer 615 Exposition Blvd #6, New Orleans, LA 70118, USA Michael B. Timmons Professor, Department of Biological and Environmental Engineering J.J. Bisogni Professor, School of Civil and Environmental Engineering Cornell University, Ithaca, NY 14853, USA Many of the current greenhouse based intensive marine shrimp production systems have used photoautotrophic algae systems to control inorganic nitrogen accumulation. A second strategy of nitrogen control used in some indoor intensive recirculating production systems is using large fixed-film bioreactors. This option utilizes chemosynthetic autotrophic bacteria, Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) for the nitrification of ammonia-n to nitrite-n and finally to nitrate-n. Recently, zero-exchange management systems have been developed which encourage heterotrophic bacterial growth. These systems have been promoted for the intensive production of marine shrimp in both ponds and raceways. In this system, heterotrophic bacterial growth is stimulated through the addition of carbonaceous substrate. At high carbon to nitrogen (C/N) feed ratios, heterotrophic bacteria will assimilate ammonia-n directly from the water and produce cellular protein. A review of the literature on zero-exchange systems yielded little description of the pathways of ammonia removal employed and whether the removal was fundamentally photoautotrophic, autotrophic or heterotrophic bacterial based or in reality some mixture of the three. The fundamental stoichiometry of the three pathways is reviewed here and the impact on water quality for each summarized. By keeping in mind the fundamentals of the three predominant pathways for ammonia-n assimilation in aquaculture production systems, it is possible to track which pathway is dominating at critical times during startup and production phases. In addition, by manipulating key water quality parameters and ambient conditions, it becomes possible to guide a system towards a specific treatment process. For example, heterotrophic systems can be selected for by adjusting the carbon/nitrogen ratio of the feed or through supplemental addition of carbon in the form of carbohydrates, e.g., sugar. These pathway indicators were then examined in a series of replicate research trials conducted at the Conservation Fund s Freshwater Institute examining water quality in juvenile marine shrimp, Penaeus vannamei, production tanks. 1

2 1.0 Introduction The economic success of an intensive production system for marine shrimp primarily rests on the ability to produce shrimp with an acceptable feed conversion ratio and adequate growth rate. For numerous reasons, most importantly biosecurity, marine shrimp production is moving towards intensive recirculating systems. Currently, the strategy most commonly used for controlling ammonia-n and nitrite-n in recirculating systems is the use of large fixed-film bioreactors that rely on the nitrification of ammonia-n to nitrate-n by Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB), bacteria. Although a proven and accepted technology, such technology is economically unattractive for marine shrimp production because of the large upfront capital costs and ongoing operational costs for pumping and maintenance. More recently, zero-exchange management systems have been developed for large-scale pond production, where carbonaceous substrate is added to the systems to support microbial metabolism (Avnimelech 1999, McIntosh 1999). 2.0 Background The three nitrogen conversion pathways traditionally used for the removal of ammonia-n in marine shrimp aquaculture systems are (1) the conversion via photoautotrophic algae to algal biomass, (2) via autotrophic bacterial to nitrate nitrogen, (3) metabolism by heterotrophic bacterial directly to microbial biomass. In order to fully understand these three conversion process, it is helpful to look at the stoichiometry involved. This, in turn, helps to conceptualize the impact of each pathway on specific water quality parameters. 2.1 Stoichiometry Photoautotrophic systems One of the most commonly used pathways for ammonia removal is through the use of greenwater systems, which use the high nutrient content of ponds, tanks, or raceways, outside or inside a greenhouse to grow algae at high density. The algae use either ammonia-n or nitrate-n and carbon dioxide as its carbon source to biosynthesize biomass. The biosynthesis of saltwater algae can be described by the following stoichiometric relationships (Stumm and Morgan, 1996): for ammonia as the nitrogen source, 16NH CO 2 + HPO H 2 O + 14 HCO 3 - C 106 H 263 O 110 N 16 P O 2 (1) or, for nitrate as the nitrogen source, 16 NO CO 2 + HPO H 2 O C 106 H 263 O 110 N 16 P O HCO 3 - (2) where C 106 H 263 O 110 N 16 P represents the stoichiometric formula for algae in seawater. Both reactions are endothermic, i.e., requires energy input, which for algae would be supplied by solar energy. Ammonia is preferentially taken up by algal phytoplankton over nitrate. Respiration occurs as the reverse of the above equations. 2

3 The following water quality impacts of photosynthesis need to be noted: photosynthesis is a one step process for both reactions, the C/N ratio of algal biomass is the same, 5.69 g C/ g N the net production of biomass is also the same, g VSS/ g N VSS consist of 36% C and 6.3% N both reactions utilize CO 2 as their primary carbon source both reactions metabolize only a small quantity of phosphorus, 0.14 g P / g N alkalinity is consumed when ammonia is the nitrogen source, 3.13 g (as CaCO 3 ) Alk/ g N alkalinity is produced when nitrate is the nitrogen source, 4.02 g (as CaCO 3 ) Alk/ g N ph increases due to consumption of carbon dioxide both reactions generate oxygen generation times for algal biomass range from 1 to 2 days at night or during low light levels, respiration dominates and the stoichometeric relations are reversed, i.e., ph decreases, oxygen is consumed, carbon dioxide is released. Obviously, one of the most important requirements for photoautotrophic systems is sunlight. In outdoor ponds, this would seem not to be a problem. However, depending upon location, weather conditions can yield extended periods with low light levels due to heavy overcast. In addition, high levels of suspended solids in the water column or high turbidity are often created in shrimp growout ponds by the activity of the shrimp on the bottom searching for food particles, i.e., bioturbation. Indoor systems have also experienced problems related to a lack of adequate light energy caused by reduced light transmissivity through greenhouse covers, which significantly reduce photoactive radiation levels, excessive foam that shade the water surface (Brune et al., 2004), and high turbidity due to excessive heterotrophic bacterial growth, which prevents sunlight penetration into the water column. 2.2 Stoichiometry Autotrophic Bacteria There are two phylogenetically distinct groups of bacteria that collectively perform nitrification. These two groups are generally categorized as chemosynthetic autotrophic bacteria because they derive their energy from inorganic compounds as opposed to heterotrophic bacteria that derive their energy from organic compounds (Hagopian and Riley, 1998). Ammonia Oxidizing Bacteria (AOB) obtain their energy by catabolizing ammonia to nitrite and include bacteria of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio. Nitrite Oxidizing Bacteria (NOB) oxidizes nitrite to nitrate, and include bacteria of the genera Nitrobacter, Nitrococcus, Nitrospira, and Nitrospina. Nitrifying bacteria are primarily obligate autotrophs, which consume inorganic carbon (in the form of alkalinity) as their primary carbon source, and obligate aerobes, which require oxygen to grow (Hagopian and Riley, 1998). Nitrification is a two step process, where ammonia is first oxidized to nitrite and then nitrite is oxidized to nitrate. The two steps are normally carried out sequentially, and 3

4 since the first step has a lower kinetic reaction rate than the second step, the overall kinetics is usually controlled by ammonia oxidation. Thus, normally there is no appreciable amount of nitrite accumulation. Equations 4 and 5 show the basic chemical conversions occurring during oxidation by AOB and NOB organisms (Metcalf & Eddy, 1991), where C 5 H 7 O 2 N represents the chemical formulation for bacterial biomass. AOB: 55 NH O HCO 3 - NOB: 400 NO NH O H 2 CO 3 + HCO 3-54 NO C 5 H 7 O 2 N + 57 H 2 O H 2 CO 3 (4) 400 NO C 5 H 7 O 2 N + 3 H 2 O (5) The overall removal process can be described as (Ebeling et al., 2006): NH O HCO C 5 H 7 O 2 N NO H 2 O CO 2 (6) The following water quality impacts of nitrification need to be noted: nitrification is a two step process, ammonia to nitrite to nitrate the second step does not start until the first step is well underway, i.e., nitrite conversion to nitrate the C/N ratio of nitrifying bacterial biomass is 4.29 g C/ g N net production of biomass is very small, 0.20 g VSS/ g N VSS consist of 53.1% C and 12.4% N the reaction utilizes alkalinity as the primary carbon source, 7.05 g (as CaCO 3 ) Alk / g N ph of the system drops as the alkalinity is consumed and carbon dioxide is produced actual carbon requirement is 1.69 g C / g N oxygen requirement is 4.18 g O 2 / g N carbon dioxide production is, 5.85 g CO 2 / g N generation time for biomass is very slow on the order of 2 to 5 days One of the most important characteristics of the autotrophic nitrification process is the extremely small amounts of bacterial biomass generated. Second because of the relatively slow maximum growth rates for nitrifiers, it becomes very easy to wash-out the nitrifying bacteria in a suspended-growth process, e.g., when suspended solids are removed to maintain water quality. This is particularly true if there is no biosolids recycling that returns some of the bacteria back into the culture system. Finally, these nitrification processes result in a large amount of alkalinity consumed (7.05 g (as CaCO 3 )/g TAN) and high levels of carbon dioxide produced (5.85 g/g TAN). This can be a significant problem requiring the addition of alkalinity to maintain an adequate source of alkalinity (100 to 200 mg/l as CaCO 3 ) for systems with limited water exchange. If alkalinity consumption is not compensated for by supplementation, ph will drop. Lowering ph will result in an inorganic carbon species shift in the water carbon balance from bicarbonate to dissolved carbon dioxide. This increase in dissolved carbon dioxide could affect some aquaculture species and in some cases be lethal. Although CO 2 4

5 concentration can be controlled with gas stripping towers, significant energy is required for pumping both the water and air through these systems. 2.3 Stoichiometry Heterotrophic Bacteria Recently, a new shrimp production strategy has emerged called intensive zero exchange systems. Removal of ammonia-n by heterotrophic bacteria is a single step process, where the ammonia-n is assimilated into bacterial biomass (Ebeling et al., 2006). The stoichiometry is shown in Equation 7, where C 5 H 7 O 2 N again represents the chemical formulation for bacterial biomass and the carbon source is represented as a simple carbohydrate, i.e., glucose, C 6 H 12 O 6. NH C 6 H 12 O 6 + HCO O 2 C 5 H 7 O 2 N H 2 O CO 2 (7) The following water quality impacts of heterotrophic bacteria need to be noted: heterotrophic conversion is a one step process the C/N ratio of bacterial biomass is the same as nitrifiers, 4.29 g C/ g N net production of biomass is 8.07 g VSS/ g N VSS consist of 53.1% C and 12.4% N the reaction utilizes carbon from carbohydrate as the primary carbon source; 6.07 g C /g N or g carbohydrate / g N the alkalinity is reduced, 3.57 g ALK / g N oxygen requirement is 4.71 g O 2 / g N carbon dioxide is produced; 9.65 g CO 2 / g N generation times for biomass are very fast with half reactions rates on the order of hours as opposed to days for autotrophs. Several aspects are important in the overall heterotrophic bacterial reaction. Paramount is the large amount of bacterial biomass produced by this reaction, compared to the autotrophic reaction, 8.07 g VSS /g N versus 0.20 g VSS/g N. This translates into extremely high growth rates and substantial quantities of total solids produced. Some form of solids management is required to remove excess TSS production. A second issue is the modest amount of alkalinity consumed as the carbon source (3.57 g/g TAN) and the resulting high levels of carbon dioxide produced (9.65 g/g TAN). As pointed out above,, this could be a problem requiring the addition of carbonate to maintain reasonable alkalinity (100 to 200 mg/l as CaCO 3 ), especially for systems with limited water exchange. As a result, zero-exchange production systems that rely on suspended or attached heterotrophic bacteria should see a slight decrease in alkalinity, large suspended solids production, and high CO 2 levels. Most importantly, theoretically there should be no production of nitrite-n, or nitrate-n in a purely heterotrophic system. Generally, some form of organic carbon supplementation is required to develop a pure heterotrophic system. This is because there is usually insufficient carbon available from the feed alone. For example a feed with 35% protein has sufficient available organic carbon to sequester only 36% of the ammonia-n by the heterotrophic bacteria (Ebeling, et al., 2006). The remaining nitrogen is available to the autotrophic bacterial population. 5

6 Thus for systems without organic carbon supplementation, a combination of heterotrophic and autotrophic bacterial systems will develop at low feed rates. At higher feed rates, the harvesting of suspended solids can actually wash-out the autotrophic bacteria due to their slow growth rates. This could result in ammonia spikes in the water column. Table 1 presents a summary of water quality characteristics for the three pathways, photoautotrophic, autotrophic and heterotrophic bacteria. The arrows indicate in which direction the concentration of the water quality parameter will normally move during the initial system start-up and during long-term operation. For example, ammonia-n will usually decrease as it is consumed by algal biomass in a photoautotrophic process. In contrast, a system based on autotrophic bacteria might initially start with increasing ammonia-n concentrations and then see a dynamic drop as the AOB kick-in. At that time, the nitrite-n concentration will increase until it too drops off when the NOB kickin. Finally, the nitrate-n concentration will slowly increase until it reaches equilibrium with either flushing rates or incidental denitrification in anoxic regions in the system. As another example, for heterotrophic systems, since ammonia-n is converted directly into bacterial biomass, there should be no increase in nitrite- or nitrate-n. Table 1. Summary table of the impacts on water quality for the three pathways for ammonia-n removal. Photoautotrophic Autotrophic Heterotrophic Start-up Production Start-up Production Start-up Production NH 4 + -N then NO 2 - -N then NO 3 - -N ph (Light) or (Dark) Alkalinity (NH 4 + -N) or (NO 3 - -N) VSS O 2 CO 2 (Light) or (Dark) TOC TN growth rate fast slow very fast stable (low concentration) increasing with time decreasing with time 6

7 3. Materials and Methods 3.1. Experimental method Triplicate studies of autotrophic, heterotrophic, and photoautotrophic systems were conducted using nine circular, flat bottom insulated fiberglass tanks of approximately 470 L. In each case, ammonia-n as ammonia chloride (7.4 g NH 4 Cl) was added along with alkalinity as sodium bicarbonate and for the heterotrophic systems, supplemental carbon as sucrose based on the stoichiometry presented above. This amounted to 23.4 g NaHCO 3 for the autotrophic systems, 10.4 g NaHCO 3 for the photoautotrophic systems and 11.8 g NaHCO 3 and 72.2 g sucrose sugar for the heterotrophic systems. Freshwater was added daily as needed to make up for evaporation or spillage losses. Each tank was equipped with a circular airline along the bottom outside perimeter using a weighted diffuser hose. In addition, two 300-W submersible aquarium heaters with built-in temperature controllers were installed in each tank, which maintained the water temperature at approximately 30º C. The two autotrophic and the two heterotrophic tanks were covered with shade cloth to help minimize algae growth. 3.2 Water Quality Analysis Daily grab samples were taken each morning and filtered through 8-12 µm filter paper with the filtrate then used to determine dissolved constituent concentrations of total ammonia-n, nitrite-n, nitrate-n, ph, and alkalinity. Standard methods were routinely used and, where appropriate, primary standards were analyzed along with the samples for quality assurance. 4. Results Only three water quality parameters are reviewed and discussed because they best typify the differences among the three different microbial systems. These parameters are nitrite- N, nitrate-n, and alkalinity. Figure 1 shows mean nitrite-n in the three replicated systems over the 10-week research period. The three autotrophic tanks demonstrate the classic delayed increase in nitrite-n concentration, a peak and then a rapid decline to very low values as the biomass of NOB increases. This long acclimation phase for the autotrophic bacteria is mainly due to the slow growth rates of the NOB bacteria and the growth inhibition at high ammonia-n concentrations. In contrast, both the heterotrophic and the photoautotrophic tanks show very low concentrations of nitrite-n over the entire research period, since there are stoichiometrically no pathways for nitrite. 7

8 50 Nitrite-N (mg-n/l) Autotrophic Heterotrophic Photoautotrophic Ammonia Addition Days Figure 1. Mean nitrite-n concentrations in three replicate tanks over the 10-week study period with addition of ammonia-n, sodium bicarbonate and carbon supplementation in the heterotrophic tanks Nitrate-N (mg/l-n) Autotrophic Heterotrophic Photoautotrophic Ammonia Addition Days Figure 2. Mean nitrate-n concentrations in three replicate tanks over the 10-weekstudy period with addition of ammonia-n, sodium bicarbonate and carbon supplementation in the heterotrophic tanks. Figure 2 shows the mean nitrate-n concentrations in the three systems over the 10-week study period. Again, the increase in nitrate-n is seen in the autotrophic systems as the nitrite-n is oxidized into nitrate-n. The fall off in nitrate-n concentration in the autotrophic systems after Day 37 of the research period is probably due to denitrification occurring in the tanks. This is supported by the data in Figure 3, which shows mean alkalinity for the three systems. As can be seen, alkalinity increases from Day 37 until Day 45 and then decreases again. Both the photoautotrophic and heterotrophic systems 8

9 show a relatively constant alkalinity concentration suggesting that sufficient alkalinity was being added to satisfy the stoichiometric demand. 200 Autotrophic Heterotrophic Photoautotrophic Ammonia Addition 180 Alkalinity (mg/l) Days Figure 3. Mean alkalinity as CaCO 3 in three replicate tanks over the 10-week study period with addition of ammonia-n, sodium bicarbonate and carbon supplementation in the heterotrophic tanks. Conclusions By frequently monitoring the water quality in the production systems, it is possible to characterize the type of removal process that is dominating removal of ammonia-n. Things to look for during the start-up and production phase of an autotrophic nitrification process: initial increase, peak and the fall off in ammonia-n concentration as nitrification by autotrophic AOB bacteria begins to convert ammonia-n to nitrite-n, rapid increase in nitrite-n concentration and then falloff as autotrophic AOB bacteria convert nitrite-n to nitrate-n, increase in nitrate-n, over time a decrease in alkalinity and probably ph, very slow bacterial growth rate, thus little increase in TSS. Things to look for in a photoautotrophic nitrification process: ammonia-n concentration remains relatively constant, at moderate levels, very little nitrite-n or nitrate-n is generated, alkalinity is consumed when ammonia-n is consumed and produced when nitrate is the nitrogen source ph remains relatively constant, substantial quantities of dissolved oxygen are generated, relatively rapid growth rate of the algal biomass. 9

10 Things to look for in a heterotrophic nitrification process: ammonia-n concentration remains relatively constant, at moderate levels, very little nitrite-n or nitrate-n is generated, heterotrophic bacteria utilize carbon from the feed or supplemental sources, ph of the system remains relatively constant, carbon dioxide is produced, heterotrophic bacteria out compete autotrophic bacteria (how can you see this?) Thus similar to reading a medical chart, one can determine the overall state of health of a nitrification system, determine the dominant N- removal pathway and, if necessary, take corrective action if the system deviates from the desired pathway. References APHA Standard Methods for the Examination of Water and Wastewater. 19th edition, American Public Health Association, New York. Avnimelech, Y Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture 176: Brune, D.E., Kirk, K., Eversole, A.G., Autotrophic intensification of pond aquaculture: shrimp production in a partitioned aquaculture system. In: Proc. 5th Intern. Conf. on Recirculating Aquaculture July 22-25, pp Ebeling, J.M., Timmons, M.B., Bisogni, J.J., Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonianitrogen in aquaculture systems. In press: Aquaculture. Hagopian, D.S., Riley, J.G., A closer look at the bacteriology of nitrification. Aquacult. Eng. 18: Metcalf and Eddy, Inc Wastewater Engineering: Treatment, Disposal, and Reuse. Third Edition. McGraw-Hill, Inc. New York. McIntosh, R.P Changing paradigms in shrimp farming: General description. Global Aquaculture Advocate 2(4/5): Stumm, W., Morgan, J.J., Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters. John Wiley & Sons, Inc., New York, 1022 pgs. Timmons, M.B., Ebeling, J.M., Wheaton, F.W., Summerfelt, S.T., Vinci. B.J., Recirculating Aquaculture Systems, 2 nd Edition. Cayuga Aqua Ventures, New York, 769 pgs. 10