Muse, Morley (2021) Characterisation of Chlorella vulgaris cell wall breakdown to improve Anaerobic Hydrolysis. PhD thesis, Victoria University.

Abstract

Microalgae can be used to polish secondary treated wastewater by removing nutrients and carbon without the addition of oxygen making it a reduced energy treatment compared to traditional extended aeration systems. The recovered microalgae in turn can be used for biofuels applications such as biogas production via anaerobic digestion and biodiesel production via lipid transesterification. Anaerobic digestion is a more feasible option due to its low energy requirement and on-site power generation ability for water utilities. Nevertheless, anaerobic digestion of microalgae has several challenges with the most difficult being the recalcitrant nature of the cell wall of most microalgae resisting microbial attack during digestion. This has resulted in low methane yields after long retention times during anaerobic digestion. Also, the rigidity of the cell walls has led to low lipids release from microalgae cells due to difficulty in extracting the intracellular cell components, affecting other biofuels production processes. Due to this, several authors have suggested a pretreatment process as a means to disrupt the cell wall structure and improve degradation of microalgae. To determine the efficiency of microalgae pretreatment, a proper quantitative technique is useful to analyse cell disruption rate. This research began by comparing different pretreatment technologies using a light microscope. The light microscope was fitted with a Neubauer haemocytometer cell counter, in addition to the use of image-J cell counting software for visual analysis to quantify cell wall disruption using Chlorella vulgaris (C.vulgaris) as the model alga. C.vulgaris was selected as the microalgae species in this project as it has been widely established as a suitable species for biofuel production and anaerobic digestion due its dominance, being a local species in Australia, higher growth rates and higher lipids content when compared to other species. Pretreatment techniques compared included thermal processes using a water bath and autoclave, mechanical processing using a high-speed homogeniser, combinations of water bath and high-speed homogeniser as well as enzymatic pretreatment using lysozyme. The results of the experiments conducted showed over 80% cell disruption using high speed homogeniser and lysozyme enzyme. Thermal pretreatment using Autoclave produced the lowest cell disruption results at 42%. The results of the combination of water bath at roiling boil for 5 minutes and 5 minutes high speed homogeniser treatment at 4,000rpm showed a 50% cell disruption rate. For the water bath thermal pretreatment alone, 20 minutes was found to be most effective producing a 65% disruption rate. However, using microscopic analysis, although effective, is time-consuming for larger cell counts, making industrial pretreatment efficiency determination a challenge. Besides, the degree of pretreatment necessary to disrupt the cell is affected by the mechanical strength of the cell wall. Currently, there is little or no test for cell wall strength measurement that is shown to impact cell wall disruption and using anaerobic digestion to quantify cell strength can be slow due to long retention times. Understanding microalgae mechanical strength would enable better selection of microalgae pretreatment methods and improve energy production from microalgae, making it a more efficient process resulting in improvements in subsequent anaerobic digestion rates. From the study, a reproducible technique using high-speed homogeniser (at speeds between 4,000rpm to 33,000rpm) to evaluate the relative cell wall strength of C.vulgaris was developed and cell disruption was determined from lipid concentration following extraction. During the technique development, several solvents including diethyl ether, hexane and dichloromethane were investigated and compared for their use in extracting broken-only algae cells from solution. Dichloromethane proved to be the most suitable solvent for wet algae lipids extraction. From the results, it was determined that significant lipids extraction was from 8,500 rpm, which was identified -1 as the critical speed with shear rate of 18,227s . The maximum shear rate at 33,000rpm was found to -1 be 70,765s . Total lipids available in the cell was calculated using a modified Bligh and dyer method of dichloromethane to methanol of 2:1. It was found that the percentage of lipids from broken only cells compared to the total lipids in the cells was about a quarter at maximum cell disruption speed of 33,000rpm. Experimental verification was conducted using chlorophyll analysis and lysozyme addition which displayed a similar trend as the lipid extraction results show that the critical speed was also observed at 8,500rpm. Lysozyme enzymatic pretreatment was investigated for cell wall disruption and its impact in anaerobic hydrolysis as previous research had shown its ability to degrade C.vulgaris cells for biofuel processes. Lysozyme was later deduced in this project to initiate cell disruption, making further cell degradation by other hydrolytic enzymes easier, leading to improved lipids extraction and better anaerobic hydrolysis. The novel technique developed will assist biofuel technologies to determine the efficiency of microalgae pretreatment and has also provided knowledge on the critical shear rate when disruption occurs. Furthermore, microalgae cells showed resistance to microbial hydrolysis during previous anaerobic digestion studies using recovered microalgae from wastewater systems. Commercial anaerobic digestion using microalgae from wastewater utilises bacteria inoculum already present in the wastewater system. The effectiveness of this, however, has been low generating low yields of methane. Researching and identifying key micro-organisms in microalgae anaerobic digestion will promote the technology and improve bio-methane yields. To achieve this, bacteria such as Escherichia coli (E.coli), Streptococcus thermophilus (S.thermophilus), Lactobacillus plantarum (L.plantarum), Acetobacter aceti (A.aceti), as well as hydrolytic enzymes such as lysozyme, amylase, cellulase, pectinase, and Aspergillus oryzae (A.oryzae) fungus were utilised in separate and combined experiments’ to show the effectiveness of microbial selection and enzymes as inoculum for degrading C.vulgaris cell wall during anaerobic hydrolysis to produce volatile fatty acids (VFA) as intermediates. The amount of VFAs produced was used as a means of experimental process efficiency and to predict potential bio-methane production. Two separate experiment batches were conducted with batch 1 having retention times of 30, 45 and 60 days. Batch 2 had a retention time of 15 days as the results from batch 1 showed optimum VFA production at 30 days retention time. From the results, optimum total VFA concentration was obtained after 15 days retention using inoculum containing mixed enzymes (lysozyme, cellulase, pectinase and amylase) at 195 mg/l. This is followed by mixed bacteria containing E.coli, S.thermophilus and L.plantarum at total VFA concentration of 161 mg/l. Literature review on the selected bacteria shows the capability of these bacteria being able to produce the selected hydrolytic enzymes. Hence, the efficiency of the bacteria in producing total VFA results close to the values obtained from the mixed enzymes. Lowest VFA production was observed in test containing A.oryzae alone at 23mg/l. The low digestion efficiency observed from the fungus has been suggested to be as a result of the possibility of no cellulose wall detected in C.vulgaris cell. Another possibility is the aerobic property of the fungus limiting its growth efficiency during digestion. To investigate this further, C.vulgaris was flocculated with A.oryzae for 24 hours as well as 72 hours and used to harvest the microalgae. The harvested C.vulgaris cells were then subjected to high-speed homogeniser treatment using the technique developed earlier before undergoing anaerobic digestion using a retention time of 13 days with sampling every two days in a separate experiment. The initial tests involving harvesting of the microalgae by flocculation shows 72 hours to produce greater flocculation efficiency with almost 100% of the cells observed to flocculate and clump together under visual observation using a motic light microscope at 400X magnification. For the cell strength tests, 72-hours flocculated algae also displayed better performance with lipids extraction efficiency of 27% more than the control containing C.vulgaris alone. The 24-hour flocculated microalgae also showed good results with 20% more lipids production compared to the control containing C.vulgaris alone. However, when the flocculated microalgae at 72-hours was investigated for anaerobic hydrolysis, the results were again low providing only 14.7mg/l of total VFA at peak observed at day 5. The results confirm the earlier findings of the possibility of the absence of cellulose in the cell wall of C.vulgaris. Hence, the use of fungus A.oryzae maybe useful only in microalgae harvesting technology and not anaerobic digestion. In addition, the project provides a detailed energy calculation of the different pretreatment strategies employed and discussed the amount of energy consumed. Thermal pretreatment was found to have a lower energy consumption at 86kJ/L feed with energy recovery for both autoclave and waterbath compared. Also, without energy recovery, thermal pretreatment was still quite low at 497 kJ/feed for autoclave and 393 kJ/L feed for waterbath. Contrarily, high speed homogeniser was found to be energy intensive at maximum speed of 33000rpm with energy consumption of 1,080 kJ/L. However, at the critical speed of 8,500 rpm, energy consumption of the high speed homogeniser was low and close to thermal pretreatment with energy recovery utilising only 88.7 kJ/L feed. Moreover, potential biomethane to be produced from the optimum anaerobic hydrolysis experiment conducted at 15-days was evaluated. An energy balance and cost analysis were documented from the various biological and enzymatic pretreatments employed. A positive energy balance was observed across the various inoculum employed. Optimum net energy production was recorded by inoculum containing mixed enzymes (lysozyme, pectinase, cellulase and amylase) at 3362 J/L feed. This is followed by mixed bacteria (E.coli, S.thermophilus and L.plantarum) inoculum with net energy production at 2769.5 J/L feed. Investigating and proposing an effective method of microalgae digestion will enable microalgae disposal from wastewater and promote energy recovery making microalgal treatment of wastewater more likely in water and waste treatment facilities.

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