CO-DIGESTION OF FOOD WASTE AND MICROALGAE TAN ZHI NAN JASON (B.Eng. (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2015 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ______________________________ Tan Zhi Nan Jason 29 Dec 2014 Co-digestion of Food Waste and Microalgae – Acknowledgements Acknowledgements I would like to take this opportunity to thank the many inspiring and helpful people who have aided me tremendously in one way or another throughout the course of my research: My supervisor, Prof Tong Yen Wah, for his valuable insights and kind understanding, and who gave me the independence and freedom to pursue my research interests. Members of Prof Tong’s group, past and present, who have provided me with valuable advice, insights, and discussions, not to mention who have injected fun and humour into the otherwise more monotonous laboratory work. In no particular order, they are, Niranjani Sankarakumar, Ingo Wolf, Louise Sugg, Sushmitha Sundar, Li Wangliang, Zhang Jingxin, Lim Jun Wei, Jonathan Lee, Zhou Danhua, Hui Xian, Lee Jeeyeon, and Ramjee Chaudhary. Special mention goes to the members of the anaerobic digestion group, who have provided me with endless inspiring and thought-provoking discussions, as well as precious advice on experimental design and technique. I would also like to specially thank my two collaborators, Ramjee Chaudhary, for working with me on cultivating microalgae, as well as Dr Lim Jun Wei, for working with me on the semi-continuous digestion of food waste. The lab officers of CREATE-SJTU, Ms Dawn Tey and Ms Mah Sook Yee, for their tireless support and help. My friends and family, for their unwavering support and understanding. And lastly, the Department of Chemical and Biomolecular Engineering for having provided the research studentship and the research opportunities and facilities that made this study possible. To everyone of you, and also to any that I have missed out, thank you. i Co-digestion of Food Waste and Microalgae – Table of Contents Table of Contents Acknowledgements i Table of Contents ii Summary iv List of Tables vi List of Figures vii List of Abbreviations ix Introduction 1.1 Background . 1.2 Objective . 1.3 Hypothesis . 1.4 Scope . 1.5 Outline of the Thesis . Literature Review 2.1 Anaerobic Digestion 2.2 Food Waste . 12 2.3 Microalgae 15 2.4 Co-digestion 21 2.5 Coupling Microalgae Cultivation to Anaerobic Digestion . 24 Materials and Methods . 26 3.1 Anaerobic Digestion Inoculum 26 3.2 Microalgae – Chlorella vulgaris . 26 ii Co-digestion of Food Waste and Microalgae – Table of Contents 3.3 Food Waste (FW) 27 3.4 Biochemical Methane Potential (BMP) Assays . 28 3.5 Microalgae Pretreatment . 30 3.6 Anaerobic Digestion Effluent 31 3.7 Microalgae Cultivation on Anaerobic Digestion Effluent 32 3.8 Characterization Methods 33 Results and Discussion 35 4.1 Co-digestion Feasibility Tests 35 4.2 Microalgae Cultivation on Anaerobic Digestion Effluent 53 Conclusion . 59 Future Work 61 Bibliography 63 Appendix A 69 Appendix B 70 iii Co-digestion of Food Waste and Microalgae – Summary Summary Integrating microalgae cultivation into anaerobic digestion (AD) for nutrient recycling and potential digester improvement is a potentially promising route for energy recovery from food waste. In this context, this study was conceived to investigate two things: 1) the co-digestion performance of food waste and Chlorella vulgaris (C. vulgaris), which includes determining the optimal mixing ratio and the effect of pretreatment of the microalgae on co-digestibility, and 2) the suitability of AD effluent as a growth medium for C. vulgaris. In the first part, food waste and microalgae were mixed and digested with ratios where 25%, 50% and 75% of food waste were replaced by microalgae on a volatile solids (VS) basis, corresponding to carbon to nitrogen ratios (C/N ratios) of 13.3, 10 and 8.07 respectively. Subsequently, the pretreatments of microalgae included thermal pretreatment at 100 0C for 45 to hr, and ultrasonic disintegration at an energy dose of 180 J/ml. In the second part, AD effluent was obtained from a 2L semicontinuous reactor digesting only food waste, and then centrifuged and the supernatant diluted to 12.5%. The resulting diluted effluent was then tested for cultivating C. vulgaris. Results showed that none of the co-digestion mixtures, pretreated or not, achieved final methane yields that were higher than digestion of food waste alone. No synergistic effects were observed, and most mixtures, especially those with pretreated microalgae, produced antagonistic effects. The reason for this was hypothesized to be long-chain fatty acid (LCFA) toxicity of microalgae, and the unsuitability of the inoculum to degrade microalgae, leading to lower methane yields for pretreated algae than untreated algae, and adverse effects on the co-digestion mixtures. iv Co-digestion of Food Waste and Microalgae – Summary Cultivation of microalgae on AD effluent on the other hand showed that AD effluent had the necessary nutrients for microalgal growth, with the growth rate comparable to that on synthetic medium. The results from this study suggested that food waste and C. vulgaris are not suitable co-substrates. Nonetheless, the successful cultivation of C. vulgaris using AD effluent showed that AD effluent could be a promising growth medium for C. vulgaris to reduce the high cost of cultivation using synthetic medium. However the low methane yields of C. vulgaris reported in this study suggested that there are possibly other inhibiting factors other than the commonly cited hard cell wall. Future work on optimising the energy recovery from microalgae is required in order to successfully couple the microalgae cultivation and anaerobic digestion process. v Co-digestion of Food Waste and Microalgae – List of Tables List of Tables Table 1. Comparison of pretreatments and methane productivity of microalgae used in anaerobic digestion Table 2. Co-digestion studies of food waste and microalgae with other substrates. Percentages in brackets indicate the best reported mixing ratio. Table 3. Characteristics of inoculum collected from Ulu Pandan WWTP over different batches. Standard deviation in brackets. Table 4. Growth conditions of Chlorella vulgaris Table 5. Characteristics of Chlorella vulgaris grown in synthetic medium. Standard deviation in brackets. Table 6. Characteristics of food waste collected from FoodClique. Standard deviation in brackets. Table 7. Experimental set-up and conditions for co-digestion batch feasibility tests. Table 8. Semi-continuous digester experimental set-up and conditions. Table 9. Characteristics of digester effluent collected one HRT after reactor start-up. Standard deviation in brackets. Table 10. Experimental set-up and conditions for microalgae cultivation on anaerobic digestion effluent Table 11. Comparison of final methane yields of Chlorella vulgaris in the literature Table 12. Absorbance at 680 nm measured on the 11th day of cultivation of C. vulgaris on synthetic medium and AD effluent. Standard deviation in brackets Table 13. Composition of synthetic medium 3N-BBM+V (Bold’s Basal Medium with 3fold Nitrogen and Vitamins; modified) vi Co-digestion of Food Waste and Microalgae – List of Figures List of Figures Figure 1. Anaerobic digestion process flow. [14] Figure 2. Chlorella vulgaris under the light microscope Figure 3. Schematic ultrastructure of C. vulgaris representing different organelles [39] Figure 4. Negatively stained cell wall microfibrils of C. vulgaris taken from [40] Figure 5. Microalgae cultivation coupled to anaerobic co-digestion of food waste and microalgae - process overview Figure 6. Methane yield of Series - Co-digestion of food waste and untreated microalgae Figure 7. Methane yield of Series - Co-digestion of food waste and heated microalgae Figure 8. Methane yield of Series – Microalgae pretreatment study on co-digestion at mixing ratio Figure 9. Comparison of final methane yields for all three series. Blue - pure food waste substrate. Green - untreated algae mixtures. Red - heated algae mixtures. Yellow - sonicated algae mixtures. Figure 10. Increase of Experimental BMP over Calculated BMP for all co-digestion mixtures. Negative values indicate that experimental BMP values were lower than calculated values. Green - untreated algae mixtures. Red - heated algae mixtures. Yellow - sonicated algae mixtures. Figure 11. Percentage COD solubilization after various pretreatments Figure 12. Ammonia concentrations after experiment in Series 1. Blank refers to bottles where no substrate was added Figure 13. Growth of C. vulgaris in synthetic medium (3N-BBM+V) and in 12.5% autoclaved AD effluent vii Co-digestion of Food Waste and Microalgae – List of Figures Figure 14. Growth of C. vulgaris in synthetic medium (3N-BBM+V) and in 12.5% nonautoclaved AD effluent Figure 15. Methane productivity and pH of the 2L semi-continuous digester with 0.5 gVS/L/day of food waste as feed viii Co-digestion of Food Waste and Microalgae – Results and Discussion nitrogen content to synthetic medium. Nevertheless, the similar exponential growth phases indicate that AD effluent is a suitable growth medium for C. vulgaris. 4.2.2 Comparison with literature Studies have found that anaerobic digestate is generally a good growth medium for microalgae, indicating that coupling microalgal cultivation to anaerobic digestion processes is promising. While studies on C. vulgaris cultivation on AD effluent are few, there are many other studies on other microalgae species that report good growth rates. For example, Fouilland et al. [88] reported that the highest maximum production rates of Scenesdesmus sp. of 115 mg DW/L/day were achieved when the microalgae was growing on 20% of its own digestate effluent (AD effluent where the feed in the AD process was the microalgae itself). Ruiz-Martinez et al. [89] reported a mean biomass productivity of 234 mg/L/day when growing mixed microalgae (Chlorophyceae and cyanobacteria) on effluent from submerged anaerobic membrane digesters digesting wastewater. Uggetti et al. [90] studied the growth of mixed microalgae (mostly Scenesdesmus sp.) on wastewater treatment plant AD effluent, and concluded that the effluent can be a good substrate for microalgal growth, with biomass yields of up to 2.6 g TS/L. As can be seen there are good precedents for the suitability of AD effluent as a growth medium for microalgae, so similar results in this work are not unexpected. Although biomass yields were not measured in this work, a comparison with synthetic medium was done, which was omitted in the references cited above. Since algal growth rates tend to be higher in artificial medium compared to real wastewater [87], it only emphasizes the suitability of AD effluent as growth medium when observing in our 55 Co-digestion of Food Waste and Microalgae – Results and Discussion results that the growth rates were comparable between cultivation in AD effluent and in synthetic medium. 4.2.3 Autoclaving and microalgae-bacteria colonies 0.600 Synthetic Medium Absorbance at 680 nm 0.500 12.5% Non-Autoclaved AD Effluent 0.400 12.5% Non-Autoclaved AD Effluent Blank (no algae) 0.300 0.200 0.100 0.000 Days Figure 14. Growth of C. vulgaris in synthetic medium (3N-BBM+V) and in 12.5% nonautoclaved AD effluent However, when the experiment was repeated without autoclaving the effluent (synthetic medium was still autoclaved to provide a control for the inoculum) microalgae could not grow. Indeed, in Figure 14 the absorbance curves of the AD effluent with and without algae added were similar, and markedly different from the control. Visually the reactors with AD effluent turned a slight cloudy white as absorbance increased. In the AD effluent reactors with algae inoculum added, clumps of algae settled at the bottom and even on the magnetic stirrers were also observed after several days, indicating that the inoculum added had died and aggregated. This was in sharp contrast to the homogenous green suspension that indicated living microalgae in the control reactors with synthetic medium. 56 Co-digestion of Food Waste and Microalgae – Results and Discussion The most likely reason for the failure of microalgae to grow in unsterilized AD effluent is probably due to the inhibitory and competitive effects of indigenous bacteria and protozoa [87]. It was observed that the non-sterilized AD effluent turned cloudy within a few days. In contrast, when autoclaved, the sterilized AD effluent blank with no algae added remained clear for more than a week, with its absorbance remaining constantly very low (Figure 13). This thus indicates that other microorganisms such as bacteria was growing very well, and probably out-competed the microalgae for resources. This thus suggests that AD effluent could be a good growth medium in terms of nutrient composition, but important aspects such as biotic components need to be taken into account when scaling up the process. This is because the autoclaving of effluent at the industrial scale is simply not economically feasible. However, the three studies cited in the previous section [88-90] did not autoclave their wastewater before cultivating microalgae in it, and yet they were able to obtain good growth rates. Perhaps the reason for this contradiction with the literature is that in this study the microalgae tested was a pure culture, while the microalgae used in these three studies were either a mixture of species within the same genus [88] or mixed microalgae and bacteria consortia [89, 90]. As such the mixed populations were likely to be more robust and thus grow better in the studies cited. 4.2.4 Implications on coupling microalgae cultivation to anaerobic co-digestion of food waste and microalgae Results show that AD effluent is a suitable medium for growing C. vulgaris, which can help reduce and offset the economic and environmental cost of purchasing nutrients for algae cultivation. However C. vulgaris was unable to grow when the effluent was not autoclaved, and was out-competed by other bacteria. The robustness of mixed 57 Co-digestion of Food Waste and Microalgae – Results and Discussion populations was proposed as a reason for cited studies not encountering the same problem. Thus, there appears to be some advantage if the microalgae being cultivated was not a pure culture, but rather a mixed population. In any case it has been observed that pure cultures were impossible to maintain in large-scale operations. In this respect, symbiotic consortia of microalgae and bacteria may be particularly interesting to ease the cultivation process. In a true coupled process as shown in Figure 5, eventually the only input into the process would be food waste. This was the rationale for using only food waste as the sole substrate in the 2L semi-continuous reactor from which AD effluent for cultivation was collected. Even though it has been shown that microalgae can grow well on it, the composition of microalgae, especially its trace mineral profile, is limited by the inputs, which is ultimately only food waste. As mentioned in the previous discussion on co-digestion and also in the literature review, the digestion of food waste alone invariably leads to inhibition. In fact this can be observed in the 2L semicontinuous reactor used in this study to provide AD effluent (Appendix B). This inhibition has been shown to be resolved by adding trace minerals [26, 28, 29]. Thus, even if co-digestion of food waste and microalgae could improve methane yield and productivity, without any additional input of trace minerals, the process may still eventually lead to failure. Nevertheless, preliminary feasibility tests on the cultivation of microalgae on AD effluent show that AD effluent is suitable, which is promising for the coupling of cultivation systems to anaerobic digestion. The major hurdle would thus be in identifying suitable microalgae strains or growth conditions that would improve the co-digestion methane yield. 58 Co-digestion of Food Waste and Microalgae – Conclusion Conclusion Co-digestion feasibility tests were conducted on food waste and Chlorella vulgaris. Mixing ratios studied were 25%, 50% and 75% of food waste replaced by microalgae. In a second series of experiments microalgae was also pretreated thermally and via ultrasound before co-digestion. Results show that none of the co-digestion mixtures, untreated or pretreated, reported a higher final methane yield than the digestion of food waste alone, nor did any report synergistic effects between the co-substrates. However co-digestion mixtures had a higher initial methane production rate than digestion of pure food waste, possibly indicating an improved and balanced nutrient profile arising from mixing the two substrates. The results also revealed that pretreatment of microalgae resulted in lowered methane productivity, contrary to expectations and to studies reported in the literature. Multiple possible causes of inhibition were discussed, with long-chain fatty acids and unsuitable inoculum being likely causes. Ammonia toxicity was both measured and estimated to be highly unlikely. It was hypothesized that mixing the substrates would result in an improved methane yield due to a better balanced nutrient profile and dilution of possible toxic compounds. This was disproved by the results. In addition, it appeared that codigestion failed to dilute any inhibitory compounds to an extent where methane yield could be improved. On a positive note, microalgae cultivation tests on AD effluent showed that AD effluent does indeed contain sufficient nutrients for microalgal growth. However this was only after the effluent was autoclaved. Unsterilized effluent was unsuitable for 59 Co-digestion of Food Waste and Microalgae – Conclusion microalgal growth, as other bacteria and fungi out-competed it for resources. This part of the hypothesis was validated. In conclusion, food waste and Chlorella vulgaris not appear to perform well together as co-substrates for anaerobic digestion. The low digestibility of microalgae probably explains to a large extent the lack of improvement of methane yields and of synergism of co-digestion. It is envisioned that there could be better synergism if the inhibitory causes of microalgae digestion were resolved, or if the species were changed to one which is more degradable. Since AD effluent can be an effective growth medium for microalgae, there is thus impetus to find an effective way to couple microalgae cultivation to anaerobic digestion. 60 Co-digestion of Food Waste and Microalgae – Future Work Future Work As discussed, LCFA inhibition was hypothesized to be the cause of inhibition in the anaerobic digestion of Chlorella vulgaris. It is thus necessary to measure the concentrations of various fatty acids and then compare them to literature. The inoculum could also be acclimatized to microalgae to see if degradability improves over time. Additionally, more co-digestion ratios could be tested, for example ratios with much less microalgae, eg. 95% FW/5% algae, or 90% FW/10% algae. Ratios could also be determined on the basis of C/N ratios. It was also noted that the growth medium dictated the resulting composition in the microalgae. It is possible that AD effluent-grown microalgae could be co-digested with food waste. This would also be a more accurate test of feasibility than using microalgae grown on synthetic medium. Once an optimal ratio is identified, a longterm study of the coupled anaerobic digestion and microalgae cultivation system could be carried out. Chlorella vulgaris was chosen predominantly because it was readily available in the laboratory, and it had robust growth characteristics. Other species such as Dunaleilla tertiolecta could also be used, although it is a marine algae species which would cause additional complications due to saline toxicity. 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Composition of synthetic medium 3N-BBM+V (Bold’s Basal Medium with 3fold Nitrogen and Vitamins; modified) Macronutrients Concentration (g/L) NaNO3 0.75 CaCl2.2H2O 0.025 MgSO4.7H2O 0.075 K2HPO4.3H2O 0.075 KH2PO4 0.175 NaCl 0.025 Micronutrients Concentration (mg/L) Na2EDTA 4.5 FeCl3.6H2O 0.582 MnCl2.4H2O 0.246 ZnCl2 0.03 CoCl2.6H2O 0.012 Na2MoO4.2H2O 0.024 Thiaminhydrochloride 1.2 Cyanocobalamin 0.01 69 Co-digestion of Food Waste and Microalgae – Appendix B Appendix B Figure 15. Methane productivity and pH of the 2L semi-continuous digester with 0.5 gVS/L/day of food waste as feed Methane productivity (ml CH4/gVS) 450 400 350 300 250 200 150 100 50 0 10 15 20 25 30 35 Days 40 45 50 266 ml of fresh inoculum added to restart the reactor Feed stopped due to reactor failure 8.3 Feed resumed pH 7.8 7.3 6.8 6.3 10 15 20 25 Days 70 30 35 40 45 50 [...]... the various studies carried out on the co- digestion of food waste and microalgae with other substrates (discussed in Chapter 2), it was hypothesized that the codigestion of food waste and C vulgaris would enhance the methane yield, due to a more balanced nutrient profile and a dilution of toxic substances potentially found in 3 Co- digestion of Food Waste and Microalgae - Introduction either substrate... rate, and that 696 000 tons of food waste are disposed of annually, which comprises 23% of the total waste disposed [6] This disposed food waste is sent directly to incineration plants, but because of the high moisture content of food waste (about 70% water), little energy recovery is expected, and there is no possibility of utilizing the nutrients in food waste As such alternatives such as anaerobic digestion. .. reports of co- digestion of food waste with Chlorella vulgaris, although there has been a study on the thermophilic (55 oC) co- digestion of Taihu algae (primarily Microcystis sp., cyanobacteria) and kitchen (food) wastes [57] The authors have found that co- digestion of Taihu algae and kitchen wastes improved biogas production, with co- digestion yielding more biogas than kitchen wastes or algae alone, and. .. and 23 Co- digestion of Food Waste and Microalgae – Literature Review micronutrients, pH and alkalinity, inhibitors and toxic compounds, biodegradable organic and dry matter [64] 2.5 Coupling Microalgae Cultivation to Anaerobic Digestion It has been highlighted that the cultivation of microalgae poses several problems: sources of carbon dioxide, and nutrients such as nitrogen and phosphorus [65] According... hypothetical coupled process would thus look like in Figure 5 24 Co- digestion of Food Waste and Microalgae – Literature Review Food Waste Anaerobic Digestion Reactor Biogas Purified Biogas Photobioreactor Liquid Effluent Algae Biomass Final Effluent Undigested Solids Figure 5 Microalgae cultivation coupled to anaerobic co- digestion of food waste and microalgae - process overview This coupled process... [47] [48] [49] Table 1 Comparison of pretreatments and methane productivity of microalgae used in anaerobic digestion 19 Co- digestion of Food Waste and Microalgae – Literature Review To counter this problem of low degradability, pretreatments have been used to great effect Table 1 shows a summary of some pretreatments reported in the literature for the anaerobic digestion of microalgae This summary... that the digestion of food waste alone invariably led to reactor failure, and that this problem could be resolved by the addition of trace elements either directly or via co- digestion [26, 28, 29] Stimulatory effects by trace 13 Co- digestion of Food Waste and Microalgae – Literature Review elements can have several reasons Many enzymes in bacteria are metal enzymes with a metal ion cofactor, and any... lipid content in microalgae During optimal growth conditions, the lipid content of C vulgaris can reach 5-40% dry weight [52] Under stress conditions this can go up to 58% [52] 20 Co- digestion of Food Waste and Microalgae – Literature Review As discussed above for food waste, lipids are converted into long-chain fatty acids as part of the degradation process These lipids can severely inhibit the digestion. .. vulgaris is capable of growing photoautotrophically, heterotrophically and mixotrophically [39] Chlorella’s ability to rapidly uptake and assimilate carbon dioxide and nutrients from waste streams and synthesize large amounts of lipids also makes it a candidate for biofuels and bioremediation [8] Figure 2 Chlorella vulgaris under the light microscope 16 Co- digestion of Food Waste and Microalgae – Literature... wall microfibrils of C vulgaris taken from [40] The rigidity of the cell wall can pose a major problem to anaerobic digestion This is discussed in the next section 17 Co- digestion of Food Waste and Microalgae – Literature Review 2.3.3 Anaerobic Digestion of Microalgae As mentioned, anaerobic digestion can be of interest if the lipid concentration is not higher than 40% Additionally, the anaerobic digestion . Microalgae cultivation coupled to anaerobic co- digestion of food waste and microalgae - process overview Figure 6. Methane yield of Series 1 - Co- digestion of food waste and untreated microalgae Figure. process. Co- digestion of Food Waste and Microalgae – List of Tables vi List of Tables Table 1. Comparison of pretreatments and methane productivity of microalgae used in anaerobic digestion. everyone of you, and also to any that I have missed out, thank you. Co- digestion of Food Waste and Microalgae – Table of Contents ii Table of Contents Acknowledgements i Table of Contents