Nitrification and denitrification by algal-bacterial biomass in a Sequential Batch Photo-bioreactor: effect of SRT

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1 Erasmus Mundus Master Course: IMETE Thesis submitted in partial fulfilment of the requirements for the joint academic degree of: International Master of Science in Environmental Technology and Engineering an Erasmus Mundus Master Course from Ghent University (Belgium), ICTP (Czech Republic), UNESCO-IHE (the Netherlands) Nitrification and denitrification by algal-bacterial biomass in a Sequential Batch Photo-bioreactor: effect of SRT Host university: MSc Thesis by Dudy Fredy Supervisor Prof. Piet Lens Mentor Dr. Peter van der Steen Delft August 2013 This thesis was elaborated and defended at the UNESCO-IHE, Delft, The Netherlands within the framework of the European Erasmus Mundus Programme Erasmus Mundus International Master of Science in Environmental Technology and Engineering " (Course N )

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3 Certification This is an unpublished MSc. thesis and is not prepared for further distribution. The author and the promoter give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to copyright laws, more specifically the source must be extensively specified when using results from this thesis. The Promoter The Author Prof. Piet Lens Dudy Fredy

4 The findings, interpretations and conclusions expressed in this study do neither necessarily reflect the views of the UNESCO-IHE Institute for Water Education, nor of the individual members of the MSc committee, nor of their respective employers.

5 for Azzania and Dyaz

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7 Abstract A post-treatment of UASB reactor s effluent by utilizing the interaction of algae and bacteria can offer lower energy consumption via photosynthetic oxygenation. To meet a stringent standard effluent it is necessary to optimize nitrogen removal. This research investigated the effects of Sludge Retention Time (SRT) on nitrification and denitrification performance. A mixed biomass culture of different species of microalgae and bacteria were inoculated in an open photo-bioreactor. The 1-L reactor was illuminated (25.9 µmol/m 2.s) and operated as a sequential batch reactor at 28ºC and ph 7.5 with 50% discharge per cycle (2 cycles per day). SRT were varied from 17 to 52 days by discharging different volume of completely mixed culture. Nitrification and denitrification of artificial wastewater (23 mgn-nh 4 /L and 200 mg COD/L) were achieved. The present study identified that SRT has only limited effect on the nitrification process and uptake by algal-bacterial biomass in a photo-bioreactor. The overall removal rates only varies from 2.1 to 2.9 mgn-nh 4 /L.h, while SRT varies from 17 to 52 days. The maximum oxygen production was occurred in SRT 17 days at a rate 0.3 mgo 2 /m 3.day. Keywords: algae, denitrification, nitrification, photo-bioreactor, photosynthetic oxygenation, SRT i

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9 Acknowledgements I would like to thank the almighty Allah for the uncountable blessing that He has bestowed upon me. My sincerest appreciations go to Dr. Peter van der Steen. Thank you so much for your guidance, input, throughout the research and thesis writing period. I am deeply grateful to Prof. Piet Lens for the advice and input throughout thesis work. Thank you to Angelica Rada and Rudatin Windaswara, for countless support, input and of course the valuable data for completing this thesis. I acknowledge Eldon Raj, Carlos Lopez Vazquez, and all the colleagues in paper writing meeting for correction and suggestions towards the finalizing of this thesis. I also thank to my colleagues in algae research: Indri Karya, Kuntarini Rahsilawati, Freweyni Tammene, and Thanh Tung Nguyen. I would like to express my appreciation for the help and assistant of the laboratory staff at UNESCO- IHE: Fred Kruis, Berend Lolkema, Peter Heering, Ferdi Battes, Frank Wiegman and Lyzette Robbemont. I also would like to thanks to Laurens Welles, Javier Sánchez Guillén and Sondos Saad who were very helpful during great months in the lab. The last and the most important, my wife Azzania. All is nothing without your support. Thank you for your pray, understanding and sacrifices. And to my son Dyaz. You are my spirit keeper. Thank you. iii

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11 Table of Contents ABSTRACT... I ACKNOWLEDGEMENTS... III 1 INTRODUCTION Background Problem Statement LITERATURE REVIEW Post-treatment options of UASB reactor s effluent Biological nitrogen removal from wastewater Nitrification Denitrification Effect of C/N ratio on nitrogen removal SRT (Solids Retention Time) Sequential Batch Reactor (SBR) Algal-bacterial consortium for wastewater treatment Microalgae Microalgae culture system Influence of environmental parameters on algal growth Effect of dark periode on nitrogen removal OBJECTIVES General objective Specific objectives MATERIAL AND METHODS Culture medium Microalgae-bacteria consortium Reactor set up and experimental design Sampling Analytical Methods Ammonium nitrogen Nitrite nitrogen Nitrate nitrogen Chlorophyll-a Total suspended solid (TSS) and volatile suspended solid (VSS) Biomass Light Absorption Calculation Solid Retention Time (SRT) Nitrogen balance Biomass composition Oxygen production by algae Statistical analysis Batch experiment RESULTS v

12 5.1 Nitrogen removal Daily nitrogenous concentration Ammonium conversion rate Nitrogen balance Chlorophyll-a concentration Suspended solids concentration Light absorption Biomass composition Solids Retention Time (SRT) Oxygen production Nitrate uptake batch experiment Microscopic observation DISCUSSION Ammonium conversion rate The effects of different operational sequences in SBR The effects of different SRT in SBR Comparison with other algal-bacterial photo-bioreactor Development of biofilm in the reactor Light regime in photobioractor Denitrification CONCLUSION AND RECOMMENDATIONS Conclusion Recommendations REFERENCES APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E... 93

13 List of tables Table 2.1: Typical average concentration of UASB reactor s effluent... 3 Table 2.2: Summary of various UASB-post treatment systems and their average level of effluent quality... 3 Table 2.3: Different operational sequences of SBR operation... 9 Table 2.4: Algal-bacterial consortium for nutrient removal from wastewater Table 4.1: Modified BG-11 medium for microalgae and bacteria culture Table 4.2: Experimental variations Table 4.3: SBR operational setting at period Table 4.4: SBR operational setting at period 2, 3 and Table 5.1: Nitrogen removal efficiency Table 5.2: Summary of ammonium conversion rate in different period Table 5.3: Nitrogen balance based on one cycle operation in SBR Table 5.4: Actual SRT calculation Table 5.5: Estimation of biomass composition Table 5.6: Estimation of oxygen production and consumption rate in the reactor Table 6.1: Comparison the result with other research vii

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15 List of figures Figure 1.1:Typical configuration of water treatment plant with UASB-Activated Sludge... 1 Figure 2.1:Typical cycles in Sequential Batch Reactor... 9 Figure 2.2:Interaction between algae and bacteria in wastewater treatment process Figure 2.3: Relation between light intensity on photoautotrophic growth of photosynthetic cells Figure 4.1: Schematic diagram of reactor set up Figure 4.2: DO concentrations profile partition in one cycle of SBR operation Figure 5.1: Daily nitrogenous concentration Figure 5.2: Ammonium conversion in one cycle of SBR operation in period 2 day Figure 5.3: Profile of chlorophyll-a concentration in different periods Figure 5.4: Average chlorophyll-a concentration in different periods Figure 5.5: Profile of SS concentration in different periods Figure 5.6: Average SS concentration in different periods Figure 5.7: Light intensity measurement points (a) and a simplified side view of the reactor (b) Figure 5.8: Estimated light penetration inside reactor Figure 5.9: Biomass compositions in each period Figure 5.10: Typical DO profile in one cycle operation in period 1(day 46) Figure 5.11: Typical DO profile in one cycle operation in period 2(day 68) Figure 5.12: Typical DO profile in one cycle operation in period 3 (day 117) Figure 5.13: Typical DO profile in one cycle operation in period 4 (day 171) Figure 5.14: Nitrate uptake performances by algae-bacteria consortium Figure 5.15: Chlorella sp. and Spirulina sp. (20x magnification) Figure 5.16: Scnedesmus sp. and Anabaena sp. (40x magnification) Figure 5.17: Algal-bacterial flocs Figure 6.1: Volumetric productivity of a photobioreactor r Ux as a function of biomass concentration C x Figure 6.2: Attached thread-former species of microalgae in reactor s wall Figure 6.3: Light fraction as function of the chlorophyll-a concentration in the reactor Figure 6.4: Typical nitrogen concentration profile within a cycle (Day 117) ix

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17 Abbreviations and symbols AOB BOD BNR C/N Chla COD DO FA FNA FSA HRAP HRT N NOB OD P PBR SBR SRT TKN TN TP TSS UASB VSS WSP WWTP Ammonia-oxidizing Bacteria Biochemical Oxygen Demand Biological Nitrogen Removal Carbon/Nitrogen Chlorophyll a Chemical Oxygen Demand Dissolved Oxygen Free Ammonia Free Nitrous Acid Free Saline Ammonia High Rate Algal Ponds Hydraulic Retention Time Nitrogen Nitrite-oxidizing Bacteria Optical Density Phosphorus Photobioreactors Sequencing Batch Reactor Solid(sludge) Retention Time Total Kjeldahl Nitrogen Total Nitrogen Total Phosphorus Total Suspended Solid Upflow Anaerobic Sludge Blanket Volatile Suspended Solid Waste Stabilization Ponds Wastewater Treatment Plant xi

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19 1 Introduction 1.1 Background High-rate anaerobic treatment system especially Upflow Anaerobic Sludge Blanket (UASB) reactor is popularly used for sewage treatment in tropical and developing countries (Seghezzo et al., 1998; Gomec, 2010). It is a sustainable technology and offers some advantages such as low cost, low energy consumption and simple operation (Chernicharo, 2006; Khan et al., 2011). However, the effluents of anaerobic reactor require a post-treatment to meet stringent discharge standards, especially the Nitrogen concentration in the effluent. Various technological options for further treating the UASB s effluent are available to achieve desired effluent quality. One of the promising options is to couple UASB with Activated Sludge System. A UASB-Activated Sludge system consists of a UASB reactor, a continues-flow aerated bioreactor and a settler as shown in Figure 1.1. Many studies reported that coupling UASB with Activated Sludge can achieve high removal of Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD) and Total Suspended Solids (TSS) (Chernicharo, 2006; Chong et al., 2012). Figure 1.1:Typical configuration of water treatment plant with UASB-Activated Sludge Source: (Chernicharo, 2006) As a variant of activated sludge system, Sequential Batch Reactor (SBR) can be coupled with UASB (Chong et al., 2012). In SBR, treatment of wastewater is carried out in various consecutive phases such as filling, reaction, settling, decant, and idle, within one reactor. SBR adds up the benefit on smaller footprint requirement. On the other hand, microalgae have been used to treat wastewater for many years in large ponds and photo-bioreactors. Microalgae have high affinity for nitrogen and phosphorous, and do not require much an organic carbon source (Boelee et al., 2012). The use of microalgae for wastewater treatment Dudy Fredy 1

20 becomes more attractive as wastewater can be a nutrient supply for microalgal biofuel production. This will bring the biofuel production more economically viable and sustainable (Boelee et al., 2011). A combination treatment of wastewater, by utilizing the interaction of algae and bacteria can offer lower energy consumption via photosynthetic aeration. Microalgae provide the oxygen necessary for aerobic bacteria to biodegrade organic pollutants and ammonium removal, Furthermore, the application of algal-bacterial system in a Sequential Batch Photo-bioreactor as a post-treatment for domestic wastewater can show a great potential. It will reduce aeration cost and land requirement, which are the major problems in developing countries. 1.2 Problem Statement Nitrification and denitrification are the main pathways for nitrogen removal in wastewater treatment. In the algal-bacterial system, nitrogen accumulation into algal biomass could also contribute to the removal process. Previous research (Karya et al., 2013) identified a stable consortium of algae (Scenedesmus) and nitrifiers that could be established in a photo-bioreactor. It was found that nitrification could takes place at rates up to 8.5 mg/l.h. The in-situ oxygen generation by algae was found to be more than sufficient for the nitrification process (Karya et al., 2013). Beside nitrification, denitrification should also be introduced in the reactor to achieve high quality of effluent. The removal of nitrogen in the SBR system can be achieved by alternating aerobic and anoxic periods during the reaction. In photo-bioreactor anoxic periods can be done by creating dark periods or dark zones. Another study (Windraswara, 2013) identified that denitrification can occur in algal-bacterial system in a photo-sbr, when no lights was applied in the anoxic periods. With a more stringent standard effluent for total nitrogen, it is necessary to establish a good nitrogen removal in Sequential Batch Photo-bioreactor. The nitrogen removal efficiency is influenced by a number of factors, such as sludge age, temperature, and carbon availability. The focus of this research therefore is to investigate the effects of sludge age or Sludge Retention Time (SRT) on nitrification and denitrification performance. 2 MSc Thesis

21 2 Literature review This chapter describes background information of post-treatment options for UASB reactor s effluent, conventional biological nitrogen removal, and algal-bacterial interactions in wastewater treatments. 2.1 Post-treatment options of UASB reactor s effluent High-rate anaerobic treatment system especially UASB reactor receives great interests for sewage treatment in tropical and developing countries (Seghezzo et al., 1998; Gomec, 2010). It offers some advantages such as low cost, low energy consumption and simple operation (Chernicharo, 2006). However, the effluents of anaerobic reactor require further post-treatment to meet stringent discharge standards. A typical average effluent concentration from UASB reactor is shown in Tabel 2.1. Table 2.1: Typical average concentration of UASB reactor s effluent Parameter Concentration (mg/l) BOD COD TSS Ammonia >15 Total N >20 Total P >4 Source: (Chernicharo, 2006) Many studies on finding the suitable post-treatment have been done. Chong et al., (2012) thoroughly reviewed the most common options for UASB reactor effluent treatments. The summary of various UASB-post treatment systems and their effluent quality can be seen in Table 2.2. The main goal of adopting a post-treatment system to treat an anaerobic effluent is to find a process that is simple in operation and maintenance, lower capital costs, and energy efficient. Table 2.2: Summary of various UASB-post treatment systems and their average level of effluent quality Total BOD COD TSS Ammonia TKN Post-treatment Unit N (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Total P (mg/l) Activated sludge (AS) no data no data no data Sequencing-batch reactor (SBR) no data 1.3 Biofilter (BF) no data no data Downflow-hanging sponge (DHS) no data 34 no data Stabilising pond (SP) no data Series Rotating-biological contactor no data 56 no data 13 8 no data no data (RBC) Constructed wetland (CW) Source : Adapted from (Chong et al., 2012) Dudy Fredy 3

22 Based on Table 2.2, the coupling of a post treatment unit to a UASB reactor is effective at removing the residual organic matter and suspended solids. But, there is still a lack of studies on the removal of nutrients (Total Nitrogen and Total Phosphorous concentrations). However for achieving a good nitrogen removal, the organic matter removal efficiency of UASB reactor should be no more than 50 70%, so that there is enough organic matter for the denitrification step (Chernicharo, 2006). 2.2 Biological nitrogen removal from wastewater Activated sludge process is one of the most common methods for treating wastewater treatment, in which nitrification and denitrification are the main pathways for nitrogen removal. Nitrification is a two steps process carried out by two different autotrophic bacterial groups. In the first step ammonium is oxidized to nitrite by ammonia oxidizing bacteria (AOB). In the second step nitrite is oxidized to nitrate by nitrite oxidizing bacteria (NOB). For completing nitrogen removal, nitrate is reduced to nitrogen gas in the denitrification process by heterotrophic bacteria Nitrification The two sequential oxidation steps in nitrification are shown below as basic stoichiometric redox reactions: NH O 2 NO 2 - NO O 2 NO H 2 O + 2H + (2.1) (2.2) By stoichiometri the oxygen requirement for conversion of ammonia to nitrate is 4.57 mgo 2 /mgn utilized. In nitrification process, dissolved oxygen (DO) concentration is the most important parameter for both AOB and NOB. Ammonia is fully oxidized to nitrate at DO concentrations higher than 1 mgo 2 /L (Campos et al., 2007). Higher dissolved oxygen concentrations do not appear to affect nitrification rates significantly, however low oxygen concentrations reduce the nitrification rate (Ekama and Wentzel, 2008). Moreover, operation at DO concentrations of 0.6 and 0.4 mgo 2 /L can cause ammonia and nitrite accumulations (Campos et al., 2007). Furthermore, nitrification releases hydrogen ions and decreases alkalinity of the mixed liquor. For each of mg FSA (Free Saline Ammonia) that is nitrified, 7.14 mg alkalinity (as CaCO 3 ) is consumed. When the alkalinity falls below about 40 mg/l as CaCO 3 then, irrespective of the carbon dioxide concentration, the ph becomes unstable and decreases to low values (Ekama and Wentzel, 2008). Hence it is important to maintain a buffer of alkalinity in the aeration tank to provide ph stability and 4 MSc Thesis

23 ensure the presence of inorganic carbon for nitrifying bacteria. The residual amount of alkalinity desired in the aeration tank after complete nitrification is at least 50 mg/l (Gerardi, 2002). It is reported that the optimum ph for AOB Nitrosomonas is 8.1 and for NOB Nitrobacter is 7.9 (Grunditz and Dalhammar, 2001). The AOB activity is 50% reduced at ph values of 6 and 10 (Jiménez et al., 2012) and NOB were strongly affected by low ph values (no activity was detected at ph 6.5). And it is also reported that no inhibition was observed at high ph values (activity was nearly the same for the ph range ) (Jiménez et al., 2011). Temperature has a significant effect on microbial nitrification activity. The rate of nitrification is reduced with decreasing temperature and, conversely, there is a significant acceleration in the rate of nitrification with increasing temperature. The optimum temperature range for nitrification is between C (Gerardi, 2002). There are some findings in the literature that light might inhibit to both AOB and NOB (Kaplan et al., 1998; Sinha and Annachhatre, 2006). Illumination with 420 lux (5,000 foot-candles) of light resulted in complete and irreversible inactivation of ammonia oxidation (Hooper and Terry, 1973). Abelliovich and Vonshak (1993) reported that light with the intensity of 3.0 x 10 3 µe/m 2.s had a strong inhibitory effect on nitrification. On that study the nitrification was done by Nitrosomonas europae in water containing a high load of organic matter, but not in water with low organic matter (Abeliovich and Vonshak, 1993). Another study stated also that NOB were more resistance to sunlight than AOB (Vanzella et al., 1989). However in general, the effect of light depends on the type of nitrifiers as well as on the environmental condition (Guerrero and Jones, 1996) Denitrification Biological denitrification process occurs under anoxic conditions, when the dissolved oxygen concentration is less than 0.5 mg/l and nitrate ions serve as electron acceptor for microorganisms. Denitrification can be also termed dissimilatory nitrite/nitrate reduction, because nitrite ions and nitrate ions, respectively, are reduced to form molecular nitrogen. There are four sequential steps in denitrification process. The first step is a reduction of nitrate (NO 3 ) to nitrite (NO 2 ), and followed by second step, where NO 2 is reduced to nitric oxide (NO). In the third step, NO is reduced to nitrous oxide (N 2 O) an obligate intermediate, some of which ultimately escapes to the atmosphere. And the last step is the reduction of N 2 O to nitrogen gas (N 2 ) (Huang et al., 2011). The overall stoichiometric equation of the conventional denitrification is shown below: Dudy Fredy 5

24 NO gCOD + H + 0.5N g biomass (2.3) On the contrary to nitrification, denitrification consumes hydrogen ions or generates alkalinity. By considering nitrate as electron acceptor, it can be shown that for every mg nitrate denitrified, there is an increase of 3.57 mg alkalinity as CaCO 3. Therefore incorporating denitrification in a nitrification system causes the net loss of alkalinity to be reduced (Ekama and Wentzel, 2008). The genera Alcaligenes, Bacillus, and Pseudomonas are the largest number of denitrifying bacteria. Most denitrifiers reduce nitrate ions via nitrite ions to molecular nitrogen without the accumulation of intermediates. However, some denitrifiers may lack of key enzyme systems to denitrify completely, then permit the production and accumulation of intermediates (Gerardi, 2002). Temperature can also affect denitrification rate. The rate is higher with increasing temperature and is inhibited at wastewater temperature below 5 C. To compensate a decreased rate at low temperature, an increased Mixed Liquor Volatile Suspended Solid (MLVSS) can enhance the number of denitrifying bacteria (Gerardi, 2002). Denitrification can occur over a wide range of ph values. It is reported that denitrification is relatively insensitive to acidity but may be slowed at low ph (Gerardi, 2002). To ensure acceptable enzymatic activity of facultative anaerobe and nitrifying bacteria, the ph in the aeration tank should be maintained at a ph value greater than 7.0. The optimal ph range for denitrification is 7.0 to 7.5 (Gerardi, 2002). Heterotrophic bacteria degrade organic carbon in order to obtain energy for cellular activity and carbon for cellular synthesis (growth and reproduction). According to Ekama and Marais (1984), under anoxic conditions, a theoretical demand of organic biodegradable substrate will be 8.67 mg COD to reduce 1 mg N-nitrate (Ekama and Wentzel, 2008). Furthermore, it is mentioned that in practical COD/N ratios required for complete denitrification are 4-15 g COD/g N, with a minimum ratio of (Kujawa and Klapwijk, 1999). The Carbon and Nitrogen (C/N) ratio of domestic wastewaters is often lower than these prescribed values, so that nitrogen removal is limited by the lack of available organic carbon source (Ryu and Lee, 2009). Sometimes an external carbon source such as acetate, methanol or ethanol is added in order to achieve denitrification for ammonia removal. As a result, biological nutrient removal through aerobic nitrification and anoxic denitrification may increase the operational cost, process complexities and energy input for aeration. 6 MSc Thesis

25 2.2.3 Effect of C/N ratio on nitrogen removal Addition of carbon substrates increases the COD/NO 3 -N ratio and may improve denitrification process. However, denitrification rate may also depend on the types of carbon and quantities of substrate added. Sodium acetate was known as effective and efficient carbon source, then methanol and glucose. It is reported that addition of sodium acetate could increase the amount of nitrate reduction even at high dosage, and improved the rate of nitrogen removal (Tam et al., 1992). Optimum C/N ratio is the ratio that leads to a maximum conversion of all nitrogen compounds to nitrogen gas with minimum organic carbon. Theoretical optimal C/N ratio is calculated to be 3.74 for denitrification system without any competition from other heterotrophs. It also depends on the characteristic of wastewater being treated. Consequently optimal C/N is not constant and must be determined experimentally (Chiu and Chung, 2003). Theoretical optimal C/N ratio may be calculated using the stoichiometric relationship for the biological denitrification process. The chemical equilibrium equation using acetic acid as carbon source was suggested as follows (Mateju et al., 1992) CH 3 COOH + NO C 5 H 7 NO 2 + HCO CO H 2 O N 2 (2.4) SRT (Solids Retention Time) SRT or sludge age is the most important operational parameter which has been used in the design, operation and control of an activated sludge systems (Ekama, 2010). The SRT is equal to the mass of solids in the reactor divided by the mass of solids leaving the system (waste activated sludge solids) per day. A successful nitrification processes in both suspended growth or attached biofilm reactors in wastewater treatment can be determined by SRT. SRT controls microorganism in the system, when the concentration of microorganisms is high, the SRT is also high (Rittmann and McCarty, 2001). And when the reactor is in a steady state, SRT is defined as the inverse of the net specific growth rate (μ -kd ). The nitrification process depends on slow growing autotrophic bacteria. This slow growth rate sets the minimal value of the SRT in the activated sludge process (Salem et al., 2006). If the SRT was shorter than the inverse of the specific growth rate (μ -1 ), this could cause a washout of nitrifying bacteria. In most Biological Nitrogen Removal (BNR) processes, a long sludge retention time (8-10 days) is required due to the slow growth rate of AOB (Ekama and Wentzel, 2008; Lee et al., 2011). On the other hand, the heterotrophs are known to have higher specific growth rates of around 4 to 13.2 day -1 Dudy Fredy 7

26 than nitrifiers which have specific growth rates of only around 0.62 to 0.92 day -1 respectively (Okabe et al., 2011). It is reported that the nitrogen removal efficiency was higher when the SRT increased (Ekama and Wentzel, 2008). Lee et al. (2008) studied the total nitrogen removal efficiency of an SBR. He observed that the TN removal efficiency could be obtained up to 66.9% at SRT 16.2 days. However as the SRT increased, the denitrification rate per mixed liquor suspended solids (MLSS) during the first anoxic period decreased significantly (Lee et al., 2008). Moreover, another study identified that the long SRT had bettered the process. It was found that at the SRT between 10.3 to 34.3 days could lessened the unfavourable effect of low temperatures and stabilized the nitrification process (Komorowska-Kaufman et al., 2006). The effect of SRT on flocs sludge characteristics in SBR was studied by Liaou, et al. (2006). It indicated that floc size was relatively stable and not subject to influence by SRT or organic loading in SBR. This study also advised that SRT should be larger than the critical SRT (9 12 days) to maintain a relatively stable microbial community for effective biomass flocculation and separation (Liao et al., 2006) In a study of algal-bacterial system in SBR, biomass productivity was generally increased as retention times decreased (Valigore et al., 2012). And longer SRT enhanced biomass settleability while shorter Hydraulic Retention Time (HRT) enhanced productivity except when washout occurred (Valigore et al., 2012) Sequential Batch Reactor (SBR) Sequential batch reactor is a modification of activated sludge process. Whereas successfully used to treat municipal and industrial wastewater (Mahvi, 2008). The difference is that the SBR performs equalization, biological treatment, and secondary clarification in a single tank/reactor using a timed control sequence. In SBR, treatment of wastewater is carried out in various consecutive phases namely filling, reaction, settling, and decant (as shown in Figure 2.1). The removal of nitrogen can be achieved by alternating aerobic and anoxic periods during the reaction (Rodríguez, Pino, et al., 2011). The duration of each cycle and the number of stages of operation depends on the type of wastewater to be treated (Rodríguez, Ramírez, et al., 2011). The advantages of operation in SBR are single-tank configuration, small foot print, easily expandable, simple operation and low capital costs (Mahvi, 2008). 8 MSc Thesis

27 Figure 2.1:Typical cycles in Sequential Batch Reactor Source: (Mahvi, 2008) Many studies have been done with the different operational sequences as shown in Table 2.3, and generally the objectives were to optimize nitrogen removal. Table 2.3: Different operational sequences of SBR operation Operational mode Operational overview Reference Intermittenly aerated SBR to treat high phenol concentration (Singh and Srivastava, 2011) Automatically controlled SBR to enhance nitrogen removal, step-feed strategy, without external carbon source (Puig et al., 2005) Automatically controlled SBR to enhance nitrogen removal, step-feed strategy, with external carbon source Guo et al. (2007) Dudy Fredy 9

28 Operational mode Operational overview Reference Automaticallay controlled (real time) SBR to remove nitrogen via nitrite, external carbon source (Wu et al., 2011) A pilot scale SBR to treat high amount of organic matter and a high amount of ammonium (Rodríguez, Ramírez, et al., 2011) Source :(Windraswara, 2013) Irvine and Bush (1979) reported that SBR is an effective biological treatment method for removing organic matter and nutrients. It could be done by distributing the influent injection and aeration periods variably and appropriately. In particular, a higher efficiency of denitrification can be achieved in the SBR method by varying the proportional distribution of the durations of the anoxic and aerobic periods during one-cycle operation. Lee et al., (2007) indicated that increasing the duration of the anoxic (II) period, which is conducive to denitrification, increases the efficiency of nitrogen removal by denitrification. 2.3 Algal-bacterial consortium for wastewater treatment The application of algal bacterial biomass for wastewater treatment now is becoming more interesting. It presents lower energy consumption via photosynthetic aeration (Muñoz et al., 2004; Safonova et al., 2004; Muñoz and Guieysse, 2006), and offers the potential use as an alternative energy source (biofuel or biogas) from its biomass. Furthermore the algal based system may also contribute to CO 2 mitigation (Muñoz and Guieysse, 2006; Subashchandrabose et al., 2011). The interactions of microalgae and ordinary heterotrophic (OHOs) bacteria in wastewater treatment process can be a symbiotic relationship. Microalgae provide the necessary O 2 for heterotropic bacteria to biodegrade organic pollutants, and the CO 2 released from bacterial respiration is used for photosynthesis. As autotrophic nitrifiers and hetereotrophic denitrifers are also present in the system, the interactions between microorganisms become more complex, as shown in Figure MSc Thesis

29 LIGHT Reclaimed Water CO2 N CO2 ALGAE O2 OHOs Wastewater Organics BIOMASS DENITRIFIERS CO2 N NO3 NITRIFIERS Figure 2.2:Interaction between algae and bacteria in wastewater treatment process Moreover, microalgae and bacteria do not limit their interactions to a simple CO 2 -O 2 interchange. Microalgae may increase bacterial activity by releasing extracellular (Wolfaardt et al., 1994). While, De-Bashan et al. (2002) reported that the presence of growth promoting bacteria Azospirillum brasilense enhanced ammonium and phosphorous removal by C. vulgaris (de-bashan et al., 2002). However microalgae may also have an unfavorable effect on bacterial activity by increasing the ph, the DO (Dissolved Oxygen) concentration, or the temperature of the cultivation broth, or by excreting inhibitory metabolites (Oswald, 2003; Schumacher et al., 2003). Or even the other way around bacteria may inhibit microalgae by producing algicidal extracellular metabolites (Fukami et al., 1997). Nevertheless, treatment of wastewater using the interaction of algal-bacterial was developed. W.J. Oswald in the year 1950s introduced such kind technology for sewage treatment (Oswald and Gotaas, 1957). Since then, many improvements have led to the use of algal-bacterial consortium in facultative ponds, high rate algal ponds (HRAP), and closed photo-bioreactors (Babu et al., 2010; Park et al., 2011; Subashchandrabose et al., 2011; Craggs et al., 2012). Table 2.4 summarizes various studies that reported results on nutrient removal by algal-bacterial consortium to treat wastewater. Dudy Fredy 11

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31 12 MSc Thesis Table 2.4: Algal-bacterial consortium for nutrient removal from wastewater Cyanobacterium/microalga Bacterium Source of wastewater Nutrients Removal Efficiency (%) Initial conc (mg/l) Reactor used Spirulina platensis Sulfate-reducing bacteria Tannery effluent Sulfate High rate algal pond (HRAP) Chlorella vulgaris Azospirillum brasilense Synthetic wastewater Ammonia Chemostat C. vulgaris Wastewater bacteria Pretreated sewage DOC Photobioreactor pilot-scale nitrogen C. vulgaris Alcaligenes sp. Coke factory wastewater NH Continuous photobioreactor phenol with sludge recirculation C. vulgaris A. brasilense Synthetic wastewater Phosphorous Inverted conical glass bioreactor nitrogen Chlorella sorokiniana Mixed bacterial culture Swine wastewater Phosphorous Tubular biofilm photobioreactor from an AS nitrogen C. sorokiniana Activated sludge bacteria Pretreated piggery wastewater TOC Glass bottle nitrogen Activated sludge Tubular biofilm photo 9 to C. sorokiniana consortium Pretreated piggery slurry TOC bioreactor nitrogen Phosphorous C. sorokiniana Activated sludge bacteria Piggery wastewater TOC Jacketed glass tank phosphorous photobioreactor + NH Euglena viridis Activated sludge bacteria Piggery wastewater TOC Jacketed glass tank phosphorous photobioreactor + NH Abbreviations: DOC=dissolved oxygen concentration; TOC=total organic carbon Source: (Subashchandrabose et al., 2011)

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33 As background information for using microalgae for wastewater treatment, the following sub-sections discuss characteristics and limiting factors for algal growth Microalgae Micro-algae may be unicellular or multicellular, and can be found in all water bodies (e,g. fresh-water, sea-water and hypersaline lakes). They are also found in soils, on plants (terrestrial and aquatic) and form symbiotic associations with a very wide range of plants and animals. Microalgae may incorporate in many types of metabolisms (i.e. autotrophic, heterotrophic, mixotrophic, photoheterotrophic) and able to have a metabolic shift as a response of changes in the environment conditions (Mata et al., 2010). Two major types of its metabolisms are photoautotrophic and heterotrophic. Photoautotrophs obtain all the elements they need from inorganic compounds and the energy for their metabolism from light. Heterotrophs obtain their material and energy needs from organic compounds synthesized by other organisms Microalgae culture system Currently, various types of bioreactors have been used for culturing algae. They have to provide suitable conditions of temperature, ph, mixing, and substrate concentration for efficient cellular metabolism. The highly controlled culture systems are known as photo-bioreactors (PBR). They can be open or closed culture systems. Open photo-bioreactors (ponds) are less expensive, more durable, and higher productivity than large closed reactors. However, ponds are more sensitive to weather conditions, evaporation, lighting and water temperature. They also are more prone to contaminations from other microalgae or bacteria, and need more land area (Mata et al., 2010). The most widely used open systems are waste stabilization ponds (WSP) and high rate algal ponds (HRAP). Closed photo-bioreactors have several advantages over open photo-bioreactors. They provide better control over growth parameters (temperature, CO 2, ph, mixing, and O 2 ), culture conditions, reduces CO 2 losses, prevent evaporation, allows higher microalgae densities, offer a more safe, higher volumetric productivities, and protected environment (Mata et al., 2010). They also are flexible for being optimized for appropriate biological and physiological characteristics of the cultivated algal species. Dudy Fredy 13

34 2.3.3 Influence of environmental parameters on algal growth The growth rate of microalgae is influenced by physical (e.g. light and temperature), chemical (e.g. availability of nutrients, carbon dioxide, ph), biological factors (e.g. competition between species, grazing by animals, virus infections) and operational factors (e.g. bioreactor design, mixing and dilution rate) (Larsdotter, 2006). Some of the most important parameters are described in below subsections Light Microalgae are phototrophs, they obtain energy from light. The light energy is converted to chemical energy in the photosynthesis, but large parts are lost as heat. Oswald (1988) reported that in outdoor ponds, more than 90% of the total incident solar energy is converted into heat and less than 10% into chemical energy. Fontes (1987) reported a conversion efficiency of sunlight energy into chemical energy of only 2% (Larsdotter, 2006). The relationship between light intensity and photoautotrophic cell growth or various cell activities is rather complex. Algal activity increases with light intensity up to µe/m 2.s (Muñoz and Guieysse, 2006), while insufficient light lowers the growth rate. Anexcess light may lead to photoinhibiton (Hsieh and Wu, 2009). Photoinhibition has therefore been observed at noon of a sunny day when irradiance can reach more than 4000 µe/m 2.s (Fuentes et al., 1999; Carvalho et al., 2011). A typical relation between light intensity and photoautotrophic growth is shown in Figure 2.3. Figure 2.3: Relation between light intensity on photoautotrophic growth of photosynthetic cells Source: (Ogbonna and Tanaka, 2000) 14 MSc Thesis

35 Absence of light or low light intensity can also lower photosynthesis efficiency, and it will leads to anaerobic conditions in the reactor (Muñoz and Guieysse, 2006). Some algae are able to grow in the dark using simple organic compounds as energy and carbon source (heterotrophic) (Perez-Garcia et al., 2010), and their cells metabolize their components to obtain maintenance energy, thus leading to a decrease in cell weight (Ogbonna and Tanaka, 2000) Temperature The growth rate of algae increases with the increased of temperature. As cited by Park et al., (2011), Soeder et al., (1985) observed that the optimal temperature for maximum algal growth rate (sufficient nutrient and light conditions) varies between algal species. The optimal temperature for many algae is between 28 and 35 C. While Harris (1978) reported that optimal temperature also varies when nutrient or light conditions are limited. The algae growth often declines when encounter to a sudden temperature change (Park et al., 2011). Temperature is also well connected with light intensity. As cited in (Muñoz and Guieysse, 2006), Abeliovich (1986) reported that excessive temperature can happen at high light intensities and high biomass concentrations, from the fact that algae convert a large fraction of the sunlight into heat Nutrients Algae nutritional requirements can be classified into macronutrients (i.e. those required at g/l concentrations) and micronutrients (i.e. those required at mg/l or μg/l concentrations). The primary essential elements for the growth of algae are carbon, nitrogen and phosphorus. Algae also need some trace elements including calcium, iron, silica, magnesium, manganese, potassium, copper, sulfur, cobalt and zinc which are also essential but rarely affect its growth in wastewater treatment (Christenson and Sims, 2011). Ammonium and nitrate are the most commonly inorganic nitrogen sources in algal media. Some of the prokaryotic algae, the blue-green algae (cyanobacteria), can also fix atmospheric N 2. The ph of the medium may decreased when ammonium is used as the only nitrogen source. In dense cultures and high temperatures, the lower ph may result into a rapid decline in growth or even death of the culture. It is also reported that high concentrations (more than one mm) ammonium may inhibit growth, especially at high temperatures (Borowitzka, n.d.). If both ammonium and nitrate are supplied the cultures generally do not take up the nitrate until the ammonium has been used up. This is because ammonium is the end product of nitrate reduction, Dudy Fredy 15

36 therefore it may cause feedback-inhibition and repression of the nitrate uptake and reduction system (Borowitzka, n.d.) - Phosphorous is another major nutrient for algae, as an inorganic form of phosphate (H 2 PO 4 and HPO 2-4 ). The take up is started by hydrolysis with the action of phosphoesterase or phosphatase enzymes The amount of inorganic phosphorus required by algae varies among algae species. The tolerance amount is normally around 50 μg/l 20 mg/l (Becker, 1994) ph ph may have a great effect on the microalgae growth rate and species composition. For example, it was found that the optimal productivity of Anabaena variabilis (cyanobacterium) was achieved at ph and decreased slightly at ph , while at ph the cell could not survive (Larsdotter, 2006). Photosynthetic assimilation (CO 2 uptake by algae) may also increase ph in the medium. The increase of ph can reach 11 or more if CO 2 is limiting and bicarbonate is used as a carbon source (Larsdotter, 2006). Nitrogen assimilation by the algae also affects ph. Assimilation of nitrate ions tend to raise the ph, but if ammonia is used as nitrogen source, the ph of the medium may decrease to as low as 3 (Becker, 1994). At high ph (above 9) free ammonia will begin to dominate over ammonium. High concentrations of ammonia may inhibit algal growth, and this toxicity is intensified at higher temperatures which may freely diffuse over membranes into the cells (Larsdotter, 2006; Markou and Georgakakis, 2011) Dissolved Oxygen (DO) The process of photosynthesis is the main source of DO in outdoor ponds. The amount of DO concentration depends on the growth rate of algae, light intensity and temperature; it reaches a maximum value during noon and then decreases as light and temperature decrease. A study using P.carterae showed that the maximum concentration of O 2 during the day was directly related to light irradiance. The highest photosynthetic rate was obtained in high light (1,900 μmol photons/m 2.s) and at low oxygen concentration (6 10 mg O 2 /L). However, high concentrations of oxygen (26 32 mg O 2 /L) at both low and high irradiances, strongly increase the degree of inhibition of photosynthesis (Moheimani and Borowitzka, 2007). 16 MSc Thesis

37 Carbon Microalgae obtain energy through photosynthesis. As indicated in the photosynthetic equation 2.4, solar energy is converted into chemical energy and this energy is then used to assimilate inorganic carbon (CO 2 ) to produce sugars and oxygen as a by-product. 6 H 2 O + 6 CO 2 + light 6 C 6 H 12 O O 2 (2.4) Inorganic (CO 2 and HCO - 3 ) and organic carbon are utilized by algae in the ponds as well as in algal culture systems. The organic carbon sources can be assimilated either chemo- or photoheterotrophically (Larsdotter, 2006). In the first case, the organic substrate is used both as the source of energy (through respiration) and as carbon source, while in the second case, light is the energy source. In several algal species e.g. Chlorella and Scenedesmus, the mode of carbon nutrition can be shifted from autotrophy to heterotrophy when the carbon source is changed (Becker, 1994). The amount of CO 2 dissolved in water varies with ph, where an addition of CO 2 results in a decreased ph. At higher ph values, for example at ph greater than 9, most of the inorganic carbon is in form of carbonate (CO 2 3 ) which cannot be assimilated by the algae. The decreased availability of CO 2 may act as a limiting factor on the algal growth, however his effect is not often evident. Aeration is one of few ways in providing carbon dioxide for algae growth (Larsdotter, 2006). A study observed in Chlorella sp. cultivation in photo-bioreactors showed that 2% aeration of CO 2 increased the total biomass productivity (Chiu et al., 2008). The CO 2 demand for microalgae can also be calculated based on its stoichiometric requirement because 1 g biomass requires 1.85 g CO 2. For example, if the growth rate of biomass is 1 g/(g.d) at biomass concentration 1 g/l, then the carbon transfer rate needed would be 1.85 g/(l.d) (Posten, 2009). 2.4 Effect of dark periode on nitrogen removal Irradiance or light is one of the major factors, which control microalgae productivity. Absence of light can cause a reduction of photosynthesis activity, which normally leads to the occurrence of anaerobic conditions in the reactor. This condition is assumed to be favored for denitrification process. In activated sludge system, denitrification has been observed to begin when DO concentration in the flocs drops below 0.6 mg/l (Schramm et al., 1999). On the other hand, there are a number of microalgae that can grow mixotrophically. Mixotrophic growth regime is a variant of the heterotrophic growth regime, where CO 2 and organic carbon are Dudy Fredy 17

38 simultaneously assimilated and both respiratory and photosynthetic metabolism operate concurrently (Perez-Garcia et al., 2011). In the absence of light, there may be a competition between microalgae and bacteria for an organic carbon source, which may result in a negative effect on the denitrification process. 18 MSc Thesis

39 3 Objectives 3.1 General objective To optimize nitrogen removal in a Sequential Batch Photo-bioreactor fed with artificial UASB effluent by variations in operational conditions. 3.2 Specific objectives 1. To study the effects of different operational sequences in an SBR cycle on the nitrification and denitrification capacity of microalgae and bacteria consortium in a Sequential Batch Photobioreactor. 2. To study the effect of SRT on the nitrification and denitrification capacity of microalgae and bacteria consortium in a Sequential Batch Photo-bioreactor. Dudy Fredy 19

40 20 MSc Thesis

41 4 Material and Methods The research was carried out in UNESCO-IHE laboratory in Delft, the Netherlands and it was a continuation of previous MSc thesis research by UNESCO-IHE student Rudatin Windaswara (Windraswara, 2013). The photo-bioreactors was operated in the SBR mode, controlled by a bioconsole system, and connected to a data logger for DO concentration and ph measurement. The work included operating the system, preparing artifical wastewater, sampling and analyzing nitrogen compounds, chlorophyll-a, VSS/TSS, COD, and light absorption. Nitrogen conversion rates are determined and nitrogen mass balances are developed. 4.1 Culture medium The artificial UASB s effluent was used as culture medium and done by modifying a microalgae medium BG-11 (Stanier et al., 1971), similar to the medium used on previous study by Windraswara (2013). The sole nitrogen source was ammonium sulphate (NH 4 ) 2 SO 4, with the concentration of nitrogen about 23 mg N-NH 4 + /L. Sodium acetate was used as the Carbon source with the concentration about 200 mg COD/L. As trace elements source, the Ogawa solution was applied to the medium for 2 ml.to avoid precipitation, MgSO 4 and CaCl 2.2H 2 0, and COD source were prepared in different reservoir flasks. The detailed compounds are shown in Table 4.1. Table 4.1: Modified BG-11 medium for microalgae and bacteria culture No Compounds Concentration (g/l) 1 C 6 H 8 O 7.H 2 0 0, (NH 4 ) 2 SO 4 0, K 2 HPO 4 0, MgSO 4 0, CaCl 2.2H 2 O 0, NaHCO 3 0, FeSO 4.7H 2 O 0, Na 2 CO 3 0, Na 2 SiO 3.5H 2 O 0, EDTA.Na 2 0, COD source (CH 3 COONa) 0,4240 Ogawa 2 solution = 2 ml Stock solution (Ogawa 2) 1 H 3 BO 4 3, MnCl 2.4H 2 O 1, ZnSO 4.7H 2 O 0, CuSO 4.5H 2 O 0, (NH 4 ) 6 Mo7O 24.4H 2 O 1, CoCl 2.6H 2 O 0, NiCl.6H 2 O 0, KI 0, EDTA.Na 2 0,0800 Dudy Fredy 21

42 4.2 Microalgae-bacteria consortium Biomass culture was consisted of algae species Scenedesmus quadricauda, Chlorella sp, Anabaena variabilis, Chlorococcus sp, and Spirulina sp, enriched with wild algae species from a canal in Delft, the Netherlands. Nitrifying and denitrifying bacteria population were taken from fresh sludge of Harnaschpolder Wastewater Treatment Plant (WWTP), Delft, the Netherlands. 4.3 Reactor set up and experimental design The experiment was conducted in 1-L cylindrical jacketed and transparent glass reactor. The temperature and ph during React phase was kept at 28 o C and 7.5, respectively. Four sets of standing lamps (Phillips, the Netherlands) were installed at four sides of the reactor (average light intensity on the surface of reactor s wall of 25.9 µmol/m 2 s). Mixing was maintained at 200 rpm during Fill and React phases, and DO concentration and ph were measured and recorded using the DO probe from Bio-console Applikon, the Netherlands. The schematic diagram of reactor set-up is shown in Figure 4.1. Figure 4.1: Schematic diagram of reactor set up Source: (Karya et al., 2013) 22 MSc Thesis

43 The reactor was operated as an open system of SBR, with two cycles per day (1 cycle in 12 hours). The experimental variations were done in four periods. The first two periods were done in different sequential operation and the other three periods were done in different SRT as described in Table 4.2. Table 4.2: Experimental variations Period Sequential operation in one cycle of SBR mode SRT (days) Influent Filling Aerobic 1 Anoxic Aerobic 2 Settling Influent and Carbon source Filling 1 Carbon source Influent and Carbon source Filling 2 Withdraw 26 4 Anoxic 1 Aerobic 1 Anoxic 2 Aerobic 2 Settling Withdraw 17 The more detailed operational setting of one cycle for each period is described in Table 3.3 and Table 3.4. Withdrawal of the effluent was 50% of the total reactor volume. The duration of each SBR phases were controlled using a set of automatic controllers from Bio-Console Applikon, Holland. Table 4.3: SBR operational setting at period 1 Time Set No Phase (min) ph Light Stirrer Remark 0 initialization 0 NA on 1 start filling on 400 ml of substrate 3 aerobic pre anoxic on 5 filling off 100 ml of COD source 6 anoxic aerobic settling 105 NA on 9 withdrawing 10 NA off 10 iddle 5 NA Total time 720 Dudy Fredy 23

44 Table 4.4: SBR operational setting at period 2, 3 and 4 Time Set No Phase Light Stirrer Remark (min) ph 0 initialization 0 NA on 1 start pre-anoxic filling anoxic off 5 aerobic on 6 pre-anoxic ,5 7 filling anoxic aerobic settling 115 NA 11 withdrawing 4 NA 12 iddle 1 NA Total time 720 off on on off 200ml of substrate + 50 ml of COD 200ml of substrate + 50 ml of COD 4.4 Sampling The samples for daily influent nitrogenous and COD concentrations were taken from the influent reservoirs at the beginning of the cycle. While the effluent samples were collected during the withdraw phase at the end of the cycle. For light absorption, TSS/VSS and chlorophyll-a analysis, the samples were collected after second filling phase completed. For all nitrogenous analysis, samples are filtered directly after the collection using 0.45 μm filter, and then stored in the refrigerator (4 o C) for the analysis. 4.5 Analytical Methods Ammonium nitrogen Based on NEN 6472 method, ammonium nitrogen concentration was measured using the spectrophotometer. Samples were filtered over a glass fiber (GF/C), pipetted into a 50 ml flask and reacted with sodium salicylate reagents and dichloroisocyanurate reagents. A series of standards of NH 4 Cl solutions were used from standard solution with known concentrations to develop a calibration curve. By using the spectrophotometer, the absorbance of each standard sample and the samples were measured at the wavelength of 655 against water with 1 cm cells between 1 to 3 hours. The results of 24 MSc Thesis

45 these standards were plotted against their known concentrations to determine the mathematical expression which further were used to determine the concentration of samples from the experiment Nitrite nitrogen Nitrite nitrogen concentration was determined according to Standard Methods for examination of water and wastewater from American Public Health Association (APHA, 1995). The analysis was using the colorimetric procedure which employs two organic reagents, namely sulfanilamide and N-(1 Naphtyl)-ethylenediamine dihydrocloride. An amino group from sulfanilamide reacts with nitrite ion - as nitrous acid which resulted in a pinkish-red azo dye. A series of NO 2 standards were used to develop a calibration curve in each analysis of the samples. Diluted samples were mixed with 2 ml of mixed reagents in 50 ml flasks after which the photometric measurement was conducted at the wavelength of 543 nm against water with 1 cm cell (between 10 minutes to 2 hours) Nitrate nitrogen Ion chromatography method using Dionex ICS-1000 is used to determine nitrate nitrogen concentration. Samples were filtered through 0.45 μm filters immediately after collection to prevent the bacteria in the sample that may change the ionic concentrations. Dilution of samples by using deionized water was needed to avoid high concentrations of nitrite nitrogen not greater than 10 mg/l. For analysis, 5 ml of samples was placed in high density polyethylene containers and washed thoroughly using de-ionized water Chlorophyll-a Chlorophyll-a concentration was determined according to Dutch Standard NEN Samples were filtered by using GF6 filter (0.45 μm porosity) and transferred to Schott GL 18 COD tubes. The chlorophyll-a was extracted using 80% (v/v) ethanol. To achieve complete pigment extraction, brief heating for about 5 minutes in a water bath at temperature of 75 o C was conducted. To promote better extraction, furthermore the tubes were shaken several times and cooled to room temperature. After centrifugation, the supernatant was analyzed in spectrophotometer at a wavelength of 665nm against 80% ethanol. For turbidity correction, measurement at a wavelength of 750 was also carried out. After obtaining the absorbance reading, 2 drops of 0.4 M HCl were added to each sample and (5 to 30) minutes later the samples were re-measured at the same wavelengths. The following equation was used to calculate the chlorophyll-a concentration: Chl-a (μg/l) = 296 * V1 * En-Ea/(Vo * p) (4.1) Dudy Fredy 25

46 Where En=Ex-Eo is the corrected absorbance of the non-acidified extract, Ea=Exa-Eoa is the corrected absorbance of the acidified extract, 296 is a correction factor on the specific absorption coefficient of Chl-a, V1 is the volume of 80% ethanol in ml, Vo is the sample volume in L and p is the cell thickness in mm Total suspended solid (TSS) and volatile suspended solid (VSS) The determination of TSS and VSS involved of drying the samples at 105 o C in an oven and combustion at 520 o C in a muffle furnace according to APHA (1995). Well mixed samples were filtered using weighed GF/C filters which had been pre-heated for 2 hours at 520 o C and stored in a desiccator. Filtered sludge solids were placed in aluminum cups and left in the oven at 105 o C for 2 hours then weighted. The dry weight (TSS) is calculated by substracting the dry weight (sample+filter) from a clean weight (filter). Samples were put back in the cups, combusted at 520 o C for 3 hours and cooled in a desiccator before weighing. The difference in weight before and after combustion represented the VSS (g/l) Biomass Light Absorption The well mixed of liquor sample from the reactor was consisted of biomass with certain chlorophyll-a concentration. Its light absorption was determined by using a spectrophotometer in a 1 cm cuvette. The initial light intensity was measured with an LI-COR LI-1400 quantum sensor. Repeating the procedure with a cuvette blanked with medium, the biomass light aborption coefficient could be determined by rearranging equation Beer-Lambert s Law as: ka = (4.2) where I is a light intensity at distance z, Io is the incident light intensity at the reactor, B is the biomass concentration, and ka is the biomass light absorption coefficient. 4.6 Calculation Solid Retention Time (SRT) The actual SRT was determined based on formula in a study done by (Valigore et al., 2012). This formula also considers the biomass concentration in wastage during discharge. The formula is shown below: 26 MSc Thesis

47 (4.3) Where: Xr biomass concentration in the reactor (mg/l) Xs biomass supernatant (mg/l) Qw waste dischare flowrate (L/day) Qs supernatant discharge flowrate (L/day) Nitrogen balance Nitrogen balance in the reactor was obtained from the overall nitrification equation by using the stoichiometric link developed by Liu and Wang in their study (Liu and Wang, 2012). The equation provides a more accurate stoichiometric link between nitrifier yield, ammonia consumption, and oxygen uptake for both steps of the nitrification process. And also considers the amounts of ammonia incorporated into the cells of ammonia oxidizers and nitrite oxidizers.the complete nitrification equation is shown below: NH NH O CO HCO C 5 H 7 O 2 N + NO H 2 O (4.4) Initial ammonium concentration Initial ammonium concentration was obtained from the measurement of influent concentration in the reservoir and multiplied it by dilution factor. Ammonium uptake by nitrifiers (AOB and NOB) Equation 4.4 shows that when 1 unit of ammonia is oxidized into nitrite then into nitrate, unit of ammonia-nitrogen would be incorporated into nitrifiers. Nitrified ammonium The highest nitrate concentration in react phase (sum of aerobic 1 and aerobic 2 for period 2 to 4) has a correlation with the ammonium nitrogen that had been oxidized (nitrified ammonium). The nitrified ammonium was calculated from the Equation 4.4 which shows that 1 mg N-NO - 3 is formed from ( ) mg N-NH + 4. Ammonium uptake by algae Since the initial ammonium nitrogen concentration is higher than the amount that had been nitrified, it means the difference was assumed as ammonium that had been uptaken by algae. And it can be assumed that there was no NO - 3 uptake by algae. Dudy Fredy 27

48 4.6.3 Biomass composition There are at least four different organisms that contributed to the total biomass in the reactor of algaebacteria consortia. Those four microorganisms namely are autotrophic nitrifiers, heterotrophic denitrifiers, ordinary heterotrophic organisms (OHO) and algae itself. The calculations of biomass production are described below: Nitrifiers biomass production Nitrifiers biomass production was obtained from Equation 4.4, where 1 mg N-NO 3 - produced 0.24 mg of biomass (C 5 H 7 O 2 N). Denitrifiers biomass production The denitrifiers organisms biomass was calculated stoichiometrically based on nitrate formed during the reaction phase. From the denitrification equation (Mateju et al., 1992) which used acetate as a carbon source, 0.55 mg of biomass (C 5 H 7 O 2 N) is produced from 1 mg N-NO - 3, as shown below: 0.819CH 3 COOH + NO C 5 H 7 NO 2 + HCO CO H 2 O N 2 (4.5) OHO biomass production Ordinary heterotrophs biomass was obtained through the stoichiometry of acetate oxidation equation. The biological reaction for acetate oxidation is adapted from Metcalf and Eddy (2003) (Henze, 2008), by combining three different half reactions of cell synthesis, electron acceptor and electron donor. The overall balanced equation is shown below: 0.125CH 3 COO NH O C 5 H 7 NO HCO CO H 2 O (4.6) Based on the measurement of influent and effluent of COD, and from COD requirement for denitrification through Equation 4.5, the COD that had been used for acetate oxidation can be known. Furthermore the OHO biomass production can be calculated. Algae biomass production The algae biomass production was calculated from the photosynthesis reaction (Mara, 2003) below: 106CO H NH HPO 4 2- C 106 H 181 O 45 N 16 P + 118O H 2 O +14H + (4.7) 28 MSc Thesis

49 DO concentration (mg/l) The biomass production was calculated stoichiometrically based on the amount of ammonium uptake by algae as described in section Oxygen production by algae The estimation of oxygen production by algae was developed from the oxygen mass balances in one cycle of SBR operation. The oxygen concentration profile of one cycle operation was divided into three phase, as illustrated in Figure Figure 4.2: DO concentrations profile partition in one cycle of SBR operation Phase I slope of phase I slope of phase III phase I phase II phase III Time (h) It started from the beginning of the cycle until the DO concentration reach near zero mg/l. It assumed that the respiration by algae was negligible. In this phase, the oxygen mass balance is shown below: (Cs-C) + r alg r nit r het (4.8) Phase II It occurred when the DO concentration reach near zero mg/l. In this phase, the oxygen mass balance is developed with assumed that the respiration by algae was negligible, and the organic material oxidation had completely finished. (Cs-C) + r alg r nit = 0 (4.9) Dudy Fredy 29

50 Phase III It occurred when the DO concentration rose until the second filling time. In this phase, the oxygen mass balance assumed that the respiration by algae was negligible, and the organic material oxidation and nitrification has completed. (Cs-C) + r alg (4.10) Where: C Cs KLa r alg r nit r het DO concentration (mg/l) Saturation DO concentration (mg/l) oxygen mass transfer coefficient (1/hour) rate of oxygen generation by algae through photosynthesis (mg/hour) rate of oxygen consumption for nitrification (mg/hour) rate of oxygen consumption for organic matter oxidation (mg/hour) The calculation procedures are described as follows: a. Equation 4.10 can be derived as follow: r alg where r = KLa*Cs + r alg k = KLa => -(1/k) ln(r - k C) = t + a (a is constant of integration) r - k C = e^(-k (t+a)), after using initial condition to evaluate a C(t) = r/k - (r/k - C₀) e^(-k t) C(t) = r/k - (1 - e^(-k t)) + Co* e^(-k t) C(t) = A - (1 - e^(-b t)) + Co* e^(-b t) (4.11) b. Determine A and B, through curve fitting of equation 4.11 with data of experiments. The curve fitting was done with online software in c. Determine the r alg from the value of A and B d. Solution of equation 4.9 and 4.8 were done in similar way. 30 MSc Thesis

51 4.7 Statistical analysis Statistical analyses were performed using free software R Studio, version Normality of the data and homogeneity of variances were determined prior to any statistical treatments with a Shapiro- Wilk s test and Q-Q plots, respectively. And the normal distribution and homogeneity of variances were not observed. The significant differences between mean values were analysed by Mann Whitney U-test, for period 1 and 2. The analysis was followed by Kruskal Wallis H-test and a Tukey s post hoc test to see the significant differences between mean values of period 2, 3, and 4. Moreover, all means were given as mean with standard deviation (number of measurements). 4.8 Batch experiment To have a better understanding on nitrate uptake by algae in the reactor, batch experiment was conducted.the same biomass culture and medium was used for the batch experiment. In the medium the only Nitrogen source was supplied from NaNO 3. Of each flask, 50 ml of the above culture was grown in 200 ml of modified BG-11 medium in duplicate to gain three treatments of 0, 9, and 18 mg/l N-NO - 3. The flasks were placed on a shaker with 140 rpm at room temperature. The lamp was positioned above the shaker with 60 µmol m -2 s -1 photon. To avoid denitrification process occurred, the light was always turned on for a period 24 hours per day. N-NO - 3 concentrations were measured daily. After the entire N-NO - 3 was diminished in the flasks, refill of N-NO - 3 was done to observe the NO - 3 uptake performance. Dudy Fredy 31

52 32 MSc Thesis

53 Nitrogenous concentration (mg/l) 5 Results 5.1 Nitrogen removal Daily nitrogenous concentration The daily nitrogenous concentration profile for all periods is shown in Figure 5.1. Nitrification was achieved completely without external aeration. A high concentration of nitrate in the effluent was observed at the end of period 1 and within period 2. It was caused by insufficient COD source for denitrification process, due to high algae growth in period 1 and COD supply failure in period 2. The explanation of why COD could be the cause is discussed in section Period 1 Period 2 Period 3 Period Inf N-NH4 Eff N-NH4 Eff N-NO2 Eff N-NO3 Days Figure 5.1: Daily nitrogenous concentration The ammonium and total nitrogen removal efficiency is shown in Table 5.1. In general, the removal efficiency for ammonium was 100% and for total nitrogen was 90%. As mentioned before due to insufficient COD in the reactor, the nitrogen removal efficiency in period 2 was low 68%. However there was no significant difference of nitrogen removal efficiency between all periods (P>0.05). Table 5.1: Nitrogen removal efficiency Ammonium Removal Period Efficiency (%) Total Nitrogen Removal Efficiency (%) Avg Std Dev Avg Std Dev Dudy Fredy 33

54 Concentration (mg/l) Ammonium conversion rate A typical nitrogeneous concentration profile in one cycle of operation in the SBR system for period 2 is shown in Figure 5.2. From the linear part of ammonium conversion curves, conversion rate constant in two different aerobic phases of the SBR operation were calculated. These rates were calculated from the slope (ka and kb) where ka denotes ammonium conversion rate constant in first aerobic phase and kb denotes ammonium conversion rate constant in the second aerobic phase ka kb Hours N-NH4 N-NO2 N-NO3 Figure 5.2: Ammonium conversion in one cycle of SBR operation in period 2 day 89 The ammonium conversion rates for all periods are summarized in Table 5.2. The statistical tests were done to see the differences of specific ammonium rate between each period. A significant difference between the specific ammonium conversion rate of period 1 and period 2 could be demonstrated (W=24, P<0.05). Table 5.2: Summary of ammonium conversion rate in different period Rate of ammonium conversion Rate of ammonium conversion (mgn- VSS Period Day (mgn-nh4/l.h) NH4/gVSS.h) ka kb average k Std Dev g/l ka kb average k Std Dev a a b a b b 3.0 Note: Different superscripts denote significant differences between periods not sharing the same superscript. Identical superscripts denote no significant difference. Values on period 1 was not included due to different variation 34 MSc Thesis

55 The specific ammonium conversion rate in period 1 was 2.4±0.2 mg/gvss.h and then decreased in period 2 to 1.1±0.1 mg/gvss.h. The specific ammonium conversion rate of period 3 (2.2±0.9 mg/gvss.h) seemed higher than period 2 (1.1±0.1 mg/gvss.h), but the difference was not significant (P>0.05). 5.2 Nitrogen balance Table 5.3demonstrates nitrogen balance per cycle in different periods after the ammonium was completely converted. The nitrified ammonium and the ammonium uptake by nitrifiers were calculated stoichiometrically by using Equation 4.4. The calculation is based on the amount of nitrate that was formed in the system during react (aerobic) phase. The ammonium uptake by algae was calculated from the difference between initial concentrations and the uptake by the nitrifiers. Table 5.3: Nitrogen balance based on one cycle operation in SBR Initial N- - Daily N-NO NH4+ 3 nitrified N-NH4+ uptake by Period Day formed concentration N-NH4+ nitrifiers (mg/d) (mg/d) (mg/d) (mg/d) N-NH4+ uptake by algae (mg/d) a b c % d avg StdDev % e avg StdDev % Assumption: - no NO 3 uptake by algae - no denitrification was occured Note: a calculated from measured influent concentration and dilution factor b taken from the highest concentration in one cycle c and d are calculated stoichiometrically from Equation 4.4 e = a-c From Table 5.3, it shows that ammonium uptake by algae is increasing from 4.8±1.1 mg/d in period 1 up to 16.6±1.3 mg/d in period 3, with the assumption that there was no nitrate uptake by algae. On the Dudy Fredy 35

56 Concentration (mg/l) Chlorophyll-a concentration (mg/l) other hand the ammonium that assimilated into nitrifiers is decreasing from 0.5±0.0 mg/d in period 1 to 0.3±0.0 mg/d in period Chlorophyll-a concentration Chlorophyll-a concentration profile of all periods in the SBR is shown in Figure 5.3. A significant difference between chlorophyll concentration of period 1 and period 2 could be demonstrated (W=210.5, P<0.01). The chlorophyll-a concentration in period 1 was 19.6±10.4 mg/l then increased to 28.1±8.1m g/l in period 2 as illustrated in Figure Period 1 Period 2 Period 3 Period Days Figure 5.3: Profile of chlorophyll-a concentration in different periods As shown in Figure 5.4, it is seemed that the chlorophyll of period 3 (7.9±3.0 m/l) was higher than with period 4 (5.1±2.1 mg/l), but there was not a significant difference (P>0.05). On the other hand if we look at the chlorophyll-a concentration profile in Figure 5.3, the increasing trend of chlorophyll concentration in period 4 can be observed Period 3 4 Figure 5.4: Average chlorophyll-a concentration in different periods 36 MSc Thesis

57 SS Concentratipn (g/l) SS concentration (g/l) 5.4 Suspended solids concentration TSS and VSS concentration profile of all periods in the SBR are shown in Figure 5.5. The statistical tests were done to see the differences of VSS average between each period. A significant difference between VSS of period 1 and period 2 could be demonstrated (W=228.5, P<0.001). The VSS in period 1 was 1.7±0.7 g/l then increased to2.3±0.6 g/l in period 2 as illustrated in Figure Period 1 Period 2 Period 3 Period Days TSS VSS Figure 5.5: Profile of SS concentration in different periods The VSS of period 3 (1.0±0.3 m/l) was not significantly different with period 4 (1.0±0.5 m/l) (P>0.05). However if we look the biomass productivity from Figure 5.6, we can see that in period 4 the productivity is higher than in period 3. Period TSS (g/l) VSS (g/l) Biomass Productivity VSS/TSS avg Std dev avg Std dev (g/l.d) avg Std dev TSS VSS Period Figure 5.6: Average SS concentration in different periods Dudy Fredy 37

58 Light Intensity ( µmol/s.m2) 5.5 Light absorption The light intensity was measured at the 12 points outer side of the reactor wall, and the average light intensity was 25.9 µmol/s.m 2, as shown in Figure 5.7a. The estimation of light penetration in the reactor was developed using the Lambert-Beer Law. This is a basis for measuring the amount of light absorption by the biomass inside the reactor. For simplification, a rectangular shape of reactor can be assumed as its side view with radius m (Figure 5.7b). The light penetration into the reactor for each period can be seen in Figure 5.8. It can be seen that for period 1 the light could only penetrate up to 1 cm from the outer wall, while in period 2, 3 and 4 the light can penetrate up to 2 cm, 4 cm, and centre of the reactor respectively. a b r=0.075 m Lamp D 14.5 cm I H G 14.5 cm Lamp C LAMP A J F LAMP B K REACTOR E REACTOR L D LAMP C 16.0 cm A C 16.0 cm Lamp A B Lamp B LAMP D Figure 5.7: Light intensity measurement points (a) and a simplified side view of the reactor (b) Reactor radius (m) Period 1 Period 2 Period 3 Period 4 Figure 5.8: Estimated light penetration inside reactor 38 MSc Thesis

59 Percentage VSS in biomass 5.6 Biomass composition The biomass compositions were estimated based on the calculated nitrogen balances in section 5.2 and a described calculation on section Figure 5.9 shows that the VSS biomass composition is consisted mostly by algae. Generally, the percentage of VSS algae in biomass increased over the period, except in period 4 (50% in period 1, 75% in period 2, 79% in period 3, and 65% in period 4). Conversely, all the microbes fraction (nitrifiers, OHOs, and denitrifiers) in biomass had a decreasing trend, except in period 4. The summary of biomass calculation is presented in Table % 80% 60% 40% 20% 0% Period 3 4 VSS algae VSS denitrifiers VSS OHO VSS nitrifiers Figure 5.9: Biomass compositions in each period 5.7 Solids Retention Time (SRT) The actual SRT was determined based on Equation 4.3, and the results are shown in Table 5.4. Table 5.4: Actual SRT calculation Period Vol reactor (L) Xr (g/l) Qw (L/day) Qs (L/day) Xs (g/l) SRT (day) Period Period Period Period Where: Xr biomass concentration in the reactor (mg/l) Xs biomass concentration in supernatant (mg/l) Qw waste discharge flowrate (L/day) Qs supernatant discharge flowrate (L/day) Dudy Fredy 39

60 40 MSc Thesis Table 5.5: Estimation of biomass composition Period Day Daily COD removed (mg/d) COD consumed for denitrification (mg/d) VSS nitrifiers (mg/d) VSS OHO (mg/d) VSS denitrifiers (mg/d) Assumption : No NO3 uptake by algae Note: f is calculated stoichiometrically from Denitrification Equation 4.5 g is calculated stoichiometrycally based on NO3 formed in Equation 4.4 h is calculated stochiometrically based on COD removed in Equation 4.6 i is calculated stoichiometrically based on NO3 formed in Equation 4.5 j is calculated stoichiometrically based on NH4 uptake in Equation 4.7 k= g + h + i + j VSS algae (mg/d) Total VSS (mg/d) Percentage of nitrifiers in VSS (%) Percentage of OHO in VSS (%) Percentage of denitrifiers in VSS (%) Percentage of algae in VSS (%) avg Std Dev avg Std Dev avg Std avg Std Dev f g h i j k

61 DO concentration (mg/l) 5.8 Oxygen production The oxygen production by algae in the reactor was estimated according to DO concentration measurement data. The typical DO profile of one cycle operation in SBR is illustrated in Figure 5.10, 5.11, 5.12, and The calculation procedure was described in section and the results are summarized in Table 5.6. Because of the same reactor set-up (stirrer velocity and medium), the oxygen mass transfer coefficient (K La ) was obtained from previous experiments done by Windaswara (2013) influent COD source Time (h) aerobic 1 anoxic aerobic 2 settling Figure 5.10: Typical DO profile in one cycle operation in period 1(day 46) Dudy Fredy 41

62 DO concentration (mg/l) DO concentration (mg/l) influent + COD influent + COD 2 0 Time (h) anoxic 1 aerobic 1 anoxic 2 aerobic 1 settling Figure 5.11: Typical DO profile in one cycle operation in period 2(day 68) influent + COD influent + COD 2 Time (h) anoxic 1 aerobic 1 anoxic 2 aerobic 2 settling Figure 5.12: Typical DO profile in one cycle operation in period 3 (day 117) 42 MSc thesis

63 DO concentration (mg/l) influent + COD influent + COD Time (h) anoxic 1 aerobic 1 anoxic 2 aerobic 2 settling Figure 5.13: Typical DO profile in one cycle operation in period 4 (day 171) The statistical tests were done to see the differences of specific oxygen production rate between each period. A significant difference between the specific oxygen production rate of period 1 and period 2 could be demonstrated (W=128, P<0.001). The specific oxygen production rate in period 1 was 4.1±0.2 mgo 2 /gvss.h and then decreased in period 2 to 1.9±0.7 mg O 2 /gvss.h. A Tukey s HSD Post-hocs test were also be done to determine which period differ from each other. Similar with the specific ammonium removal rate, it was found that even though the oxygen production rate of period 3 (6.9±1.8 mgo 2 /gvss.h) seemed higher than for period 2 (1.9±0.7 mg O 2 /gvss.h), there was not a significant difference (P>0.05). Table 5.6: Estimation of oxygen production and consumption rate in the reactor Period Oxygen production by algae (mg O2/L.h) (mg O2/gVSS.h) Oxygen consumption rate for nitrification (mg O2/L.h) (mg O2/gVSS.h) Oxygen consumption rate by OHOs (mg O2/L.h) (mg O2/gVSS.h) 1 9.7± ± ± ± ± ± ±1.4 a 1.9±0.7 a 8.7± ± ± ± ±1.7 b 6.9±1.8 a 11.2± ± ± ± ±2.4 b 18.2±14.0 b 11.6± ± ± ±175.3 Note: Different superscripts denote significant differences between periods not sharing the same superscript. Identical superscripts denote no significant difference. Values on the period 1 were not included due to different variation. Dudy Fredy 43

64 N-NO3 concentration (mg/l) 5.9 Nitrate uptake batch experiment Based on the measurement results of each one cycle operation, it is known that the nitrate concentration in settling phase was not decreasing. It means that there was no indication that algae could assimilate nitrate into its biomass. Therefore to have a better understanding on nitrate uptake by algae in the reactor, a batch experiment was conducted (see section 4.8). The batch experiment was conducted within ten days. The refill of NO 3 - was done on the seventh day. The nitrate removal rate became higher after the second addition of the NO 3 -, from mg/l.day in the first filling to mg/l.day at the second filling as shown in Figure Day 0 mg/l of N-NO3 9 mg/l N-NO3 18 mg/l N-NO3 Figure 5.14: Nitrate uptake performances by algae-bacteria consortium 5.10 Microscopic observation During the experiments, microscopic observation was done weekly. All the microalgae that were cultured from the start of the experiment still appeared in the reactor at the end of the experiments, as shown in Figure Figure 5.17 shows an algal-bacterial flocs in the photo-bioreactor. Figure 5.15: Chlorella sp. and Spirulina sp. (20x magnification) 44 MSc thesis

65 Figure 5.16: Scnedesmus sp. and Anabaena sp. (40x magnification) Figure 5.17: Algal-bacterial flocs Dudy Fredy 45

66 46 MSc thesis

67 6 Discussion 6.1 Ammonium conversion rate It is hypothesized that the ammonium conversion rate in the algal-bacterial photobioreactor is (i) proportionally correlated with dissolved oxygen concentration and light penetration into the reactor, and (ii) inversely correlated with biomass concentration. Coincide with algal-biomass assimilation the ammonium conversion can be occurred through nitrification (Muñoz et al., 2005; González et al., 2008; Karya et al., 2013), which its rate is affected by oxygen supply in the reactor. An adequate dissolved oxygen level is required to maintain a high nitrification rate (Campos et al., 2007). In photosynthetic oxygenation system, light is a basic energy source for algae to produce oxygen. To enhance photosynthesis efficiency, an appropriate light intensity should be provided in photobioreactor (Carvalho et al., 2011). This light intensity depends on its penetration which corresponds with biomass concentration. The higher biomass concentration exhibit the larger mutual shading of the cell, which then reduces the penetration depth (Grima et al., 1999). In this study ammonium conversion rates were calculated based on the slope of ammonium concentration profile in one cycle operation. It yielded zero order rates as the curves showed linear line, especially for the first two hours of aerobic phase. Moreover, the nitrification is at maximum rate (zero-order) when ammonium concentration is higher than its half saturation constant (K S,NH4 ). It is reported that half saturation constant for ammonium (K S,NH4 ) is about 1.2 mg/l (Wolf et al., 2007), so it is likely that in react phase, the ammonium removal could follow zero order reaction. However as the ammonium concentration became lower, and then the reaction is changed to a transition between zero order and first order kinetics. In the normal nitrification process, the ammonium conversion mostly follows first order kinetics reaction. From the results of the experiments, the specific ammonium conversion rate in period 1 was higher than in period 2. This could be due to the limited oxygen concentration in the reactor in period 2. In period 2, ammonium and COD source are fed simultaneously, induced the competition between OHOs and nitrifiers, whereas in period 1 such competition did not exist. On the other hand, when the SRT decreased from 52 days (period 2) to 26 days (period 3), the specific ammonium conversion rate seemed to increased, from 1.1±0.1 mg/gvss.h (period 2) and 2.2±0.9 mg/gvss.h (period 3). However there was not significant different can be observed between rate in period 2 and period 3 (P>0.05). But when SRT was decreased again to 17 days (period 4), the specific ammonium conversion rate increased to 4.7±3.0 mg/gvss.h. This may related to higher oxygen supply in the reactor during Dudy Fredy 47

68 period 4. And this is consistently supported by the results of oxygen production calculation. In all periods the oxygen production (volumetric and specific) rates have a similar trend with the specific ammonium conversion rate. For example, the specific oxygen production rate increased from 6.9±1.8 mgo 2 /gvss.h (period 3) to 18.2±14.0 mgo 2 /gvss.h (period 4). However, in this study we could not see the similar trend for volumetric ammonium conversion rate. This might be due to smaller sample size. Following the hypothesis, the ammonium conversion rate is also affected by the light penetration into the reactor and biomass concentration. The calculated estimation of light penetration inside the reactor shows the similar trend with oxygen production rate (volumetric and specific) and the specific ammonium conversion rate in all periods. Likewise, the similar trends also is shown from the inverse measured biomass concentration (TSS/VSS and Chlorophyll-a). Based on the discussion above, it can be said that the hypothesis may be true on specific ammonium conversion rate. 6.2 The effects of different operational sequences in SBR Based on a study by Wu et al (2011), the sequential operation mode in period 1 was applied, and it supported nitrification and denitrification successfully (Windraswara, 2013). However, it does not represent the actual wastewater treatment process since the influent of carbon source was fed at a different time than the ammonium source. Therefore in period 2 to 4, the sequential operation mode was changed, where the influent of ammonia and carbon source was fed simultaneously. The results of this study show that ammonium is completely removed, through nitrification and algae uptake, in all periods. And the sequential operation in period 1 resulted in higher total nitrogen removal efficiency. The total nitrogen removal efficiency was observed lower in period 2, due to the fact that the aerobic phase occurred just before the settling phase, which caused the nitrate to appear in the effluent. Based on the nitrogen concentration profile in one-cycle operation, the concentration of nitrate during the settling phase did not change, neither during the react phase. During react phase, even though ammonium concentration down to zero, but the formed nitrate concentration did not change. This shows that there was no observable nitrate uptake took place. In contrast, it is reported in literature that algae can take up the nitrate when all the ammonium has been used up (Borowitzka, n.d.). This may be because of the nitrate uptake by algae require sufficient time for acclimation and for uptake, to switch 48 MSc thesis

69 from ammonium as nitrogen supply. According to the batch experiment result, the required time to assimilate about 2 mg N-NO 3 - /L is 24 hours. This uptake rate could not be observed during the short duration of the phases. The operation in period 1 produced an increasing biomass concentration. At the end of period 1, the competition between heterotrophic microbes and algae for carbon source might have appeared and resulted in high concentration of nitrate in the effluent. For an optimal denitrification process the carbon availability should be enough. In this study, it is not only the OHOs and denitrifiers who utilize the carbon, but maybe also the algae. After double dosage of carbon was applied, it was observed that the denitrification process performed well; there were no nitrates in the effluent anymore. This confirmed that denitrification was limited by carbon source, which may have been caused by algae mixotrophic growth. They can switch their metabolism from autotrophic to heterotrophic in dark condition. It is in accordance with literature that explained Chlorella sp could grow heterotrophically and uptake acetate as carbon source (Perez-Garcia et al., 2011). 6.3 The effects of different SRT in SBR It is reported that the SRT can be effectively control the microorganism s growth in the reactor through wastage the mixed liquor (Rittmann and McCarty, 2001). The longer SRT can accommodate a secure nitrification process. Because nitrifiers are slow growing autotrophic microorganisms, need a longer time to increase its capacity for removing ammonium. Generally, the ammonium removal efficiency and nitrogen removal were not different for each SRT (P>0.05). At all periods, the ammonium was removed completely through nitrification and biomass accumulation. The percentage of ammonium removal pathway in the reactor can be seen through result of nitrogen balance calculation. Nitrification was responsible for 48% (period 2), 41% (period 3), and 59% (period 4) of ammonium removal, whereas algae biomass accumulation was responsible for 52%, 59%, and 41%, respectively. No statistical differences were observed for the nitrogen balances calculation for different periods. Based on the results, with decreasing SRT the biomass concentration (both chlorophyll-a and suspended solids) is significantly decreased from period 2 to period 3. However, the biomass concentration in period 4 (SRT 17d) was not significantly different with that in period 3 (SRT 26 d) (P>0.05). It might be that in period 4 the decreased SRT did not affect in decreased biomass but it reaches some point where the biomass started to increase again. It is probably due to better light intensity and oxygen production. Dudy Fredy 49

70 The biomass productivity in lower SRT (period 4) (0.053 gvss/l.d) was higher than in period 3(0.037 gvss/l.d). These results are in accordance with a study by Valigore et al,. (2012), who identified that a lower SRT could enhanced the biomass productivity in algal-bacterial biomass grown on primary treated wastewater. Moreover according to Janssen and Lamers (2013), the algal photobioreactor volumetric productivity (r u x) is a function of biomass concentration (C x ) and the specific growth rate of microalgae (µ), which is light-limited growth. And the relation can be plotted as shown in Figure 6.1.The photobioreactor will operate at its highest productivity at a certain biomass concentration (Cx,opt). In this research lowering biomass concentration via lowering the SRT could decrease the biomass concentration, and therefore might increase the photobioreactor productivity. Figure 6.1: Volumetric productivity of a photobioreactor r Ux as a function of biomass concentration C x Source: (Janssen and Lamers, 2013) On the other hand, from nitrifiers kinetic growth equation below: (6.1) the ammonium conversion rate (dna/dt) is proportional with biomass concentration X BA. Nitrifiers as slow growing autotrophic microorganisms need a higher biomass concentration to increase its capacity for removing ammonium. But the higher the concentration of nitrifiers, the more shading they are causing, which reduces the photosynthesis by algae and the production of oxygen in the reactor. 50 MSc thesis

71 From above discussion, it can be identified that reducing the SRT may increase oxygen production rate, and may increase ammonium conversion rate, even only a small effect. It can also be identified that reducing SRT may increase biomass productivity, but that will lead to larger mutual shading. On the other hand a long SRT can guarantee the nitrification occurred completely and produce more stable sludge. But also could require larger volume of reactor due to accumulation of fast growing heterotrophs and inert suspended solids. A decision on what SRT should be applied on photo-activated sludge should consider on an optimum biomass concentration that can provide enough oxygen for nitrification but do not lead to larger mutual shading. 6.4 Comparison with other algal-bacterial photo-bioreactor The comparison with other researches can be summarized in Table 6.1. Table 6.1: Comparison the result with other research Research /Refference Max ammonium conversion rates (mg N/L.h) Oxygen production rate (kg O2/m3.d) Biomass productivity (gvss/l.d) Ammonium removal via biomass accumulation(%) This research (Karya et al., 2013) HRAP (Muñoz and Guieysse, 2006) Enclosed Photobioreactor (Muñoz and Guieysse, 2006) (Su et al., 2011) MaB-flocs (Van Den Hende et al., 2011) The observed maximum ammonium conversion rate was 3.4 mgn-nh 4 /L.h. However, this values are lower than reported in previous study (7.7 mgn-nh 4 /L.h) (Karya et al., 2013). The low value is probably due to lower light intensity applied. And competition with hetereotrophs for oxygen due to COD source was fed in this study. The maximum observed oxygen production rate occurred in period 4 (0.30 kgo 2 /m 3.day). This value was lower if we compared with A previous study by Karya et al. (2013) (0.46 kgo 2 /m 3.day), and far below the rates that had been produced by enclosed photo-bioreactors ( kgo 2 /m 3.day) (Muñoz and Guieysse, 2006). However, the oxygen production rate in this study was similar with the oxygen Dudy Fredy 51

72 production by HRAP ( kgo 2 /m 3.day) (Muñoz and Guieysse, 2006). This lower oxygen production in this study was probably mainly caused by a lower applied light intensity. The results of nitrogen balance calculation showed that on average the ammonium removal was done via nitrification (41-77%) and algae uptake (23-59%). The effect of different SRT could not be observed due to insignificant different between periods (P>0.05). These results are in accordance with the study that used municipal wastewater, where the biomass accumulation was 44% (Su et al., 2011). Moreover, the biomass accumulation in this study is higher compared with previous study by Karya et al., (2013) about 15-19%. The biomass productivity in this study ranged between gvss/l.day. It is lower compared with microalgal-bacterial flocs (MaB-flocs) from other lab scale photo-bioreactor which has productivity around gvss/l.day (Van Den Hende et al., 2011). Based on nitrogen mass balances, the fraction of biomass was calculated. It was found that the biomass of is consisted of nitrifiers ( %), algae (48-79%) and the heteretrophs (19-45%). The low percentage of nitrifiers in the biomass is probably due to nitrification inhibition by algae, where the both compete for the same carbon and nitrogen source (Choi et al., 2010). 6.5 Development of biofilm in the reactor The development of biofilm attached on the reactor s wall became more intense after about 100 days of operation. In this study, the biofilm occurrence was considered as a negative factor. Since it reduced the light penetration into reactor and created unequal biomass concentration within the reactor. The formation of biofilm was influenced by many factors, such as extra polymeric substances (EPS) produced by bacteria and microalgae species selection (Irving and Allen, 2011). The microscopic observation was done, the species which was dominant sticking on the wall is the thread forming microalgae. This species was the filamentous algae, and it may be the Spirulina sp. (Figure 6.2). The reason why the algae have a tendency to adhere on the wall of the reactor is still unclear. It may be due to its motility for a better light catchment, or to have a better nutrient source. The coexistence with microbes which also produce EPS, might be another factor that can increase the tendency of microalgae to attach on any surfaces. 52 MSc thesis

73 The relative photic volume (ε) a b c Figure 6.2: Attached thread-former species of microalgae in reactor s wall (a=10x magnification, b= 20x magnification, and c=40x magnification) Some possible measures to reduce the tendency of biofilm formation are as follows: a. Applying higher stirring velocity without destructing the biomass. b. Microalgae species selection. c. Consideration on other reactor design for easier maintenance work, such as flat panel type of reactor. 6.6 Light regime in photobioractor The light regime inside a reactor can be characterized with a photic zone of intense light at the reactor surface and a dark zone in the interior of the reactor. A photic zone is defined as the depth at which 90% of the incoming photon flux is absorbed, and called the light penetration depth (dp) (Janssen et al., 2003). The relative photic volume (ε) is defined as the ratio of the volume of the photic zone over the reactor volume. The estimated light regime for light absorption by chlorophyll at wavelength 550 nm inside the reactor is presented in Figure Chlorophyll-a (mg/l) Figure 6.3: Light fraction as function of the chlorophyll-a concentration in the reactor As can be seen from Figure 5.2, the higher concentration of biomass it can reduce the light fraction absorbed in the reactor. The light can only penetrate into the interior reactor when the cholorophyll Dudy Fredy 53