Granulation and microbial community dynamics in the chitosan-supplemented anaerobic treatment of wastewater polluted with organic solvents

The effect of chitosan on the development of granular sludge in upflow anaerobic sludge blanket reactors (UASB) when treating wastewater polluted with the organic solvents ethanol, ethyl acetate, and 1-ethoxy-2-propanol was evaluated. Three UASB reactors were operated for 219 days at ambient temperature with an organic loading rate (OLR) of between 0.3 kg COD m−3 d−1 and 20 kg COD m−3 d−1. One reactor was operated without the addition of chitosan, while the other two were operated with the addition of chitosan doses of 2.4 mg gVSS−1 two times. The three reactors were all able to treat the OLR tested with COD removal efficiencies greater than 90%. However, the time required to reach stable operation was considerably reduced in the chitosan-assisted reactors. The development of granules in the reactors with chitosan was accelerated and granules larger than 2000 μm were only observed in these reactors. In addition, these granules exhibited better physicochemical characteristics: the mean particle diameter (540 and 613 μm) was approximately two times greater than in the control reactor (300 μm), and the settling velocities exceeded 35 m h−1. The extracellular polymeric substances (EPS) in the reactors with the chitosan was found to be higher than in the control reactor. The protein-EPS content has been correlated with the granule size. The analyses of the microbial communities, performed through denaturing gradient gel electrophoresis and high-throughput sequencing, revealed that the syntrophic microorganisms belonging to genus Geobacter and the hydrogenotrophic methanogen Methanocorpusculum labreanum were predominant in the granules. Other methanogens like Methanosaeta species were found earlier in the chitosan-assisted reactors than in the control reactor

Investigation of the microbial community structure in enhanced anaerobic sludge digestion

Sludge is an inevitable by-product in the current wastewater treatment process utilizing activated sludge technology, and its treatment and disposal could account up to 50% of WWTP’s operating costs. Optimization of the anaerobic sludge digestion to enhance volatile solids (VS) reduction and energy recovery (via methane production) is critical in the face of global challenges today. Process improvement employing biological methods (two-stage system) is preferred due to its high digestion efficiency and relatively lower operating cost and energy requirement compared to other methods. However, the underlying microbial diversity, its interactions, functions and responses to operational or environmental conditions are not clearly understood. In this study, sludge digestion enhancement efforts are complemented with microbiological molecular tools including denaturing gradient gel electrophoresis (DGGE), quantitative polymerase chain reaction (qPCR) and high throughput 454 pyrosequencing to address this research gap.Doctor of Philosoph

In-situ alkaline enhanced two-stage anaerobic digestion system for waste cooking oil and sewage sludge co-digestion

Anaerobic digestion is a promising way for resource recovery from waste cooking oil (WCO) due to its high bio-methanation potential. In-situ mild alkaline (pH 8) enhanced two-stage continuous stirred tank reactors (ALK-2-CSTRs) were implemented to explore its efficiency in co-digesting WCO and sewage sludge with stepwise increase of WCO in the co-substrates. Results demonstrate that the ALK-2-CSTRs effectively promoted methane yield from the co-substrates via promoting hydrolysis, long chain fatty acids (LCFAs) degradation and protecting methanogens from exposure to high concentration of LCFAs directly. The maximum methane yield of the ALK-2-CSTRs is 39.2% higher than that of a single stage CSTR system at the optimal feed mixture of 45:55 (WCO:SS [VS]). The thermophilic operation applied to the stage-1 of the ALK-2-CSTRs failed to improve the methane yield when the methanogenic performance was stable; while upon WCO overloaded, the elevated temperature mitigated the deterioration of methanogenesis by stimulating the bioconversion of the toxic LCFAs, especially the unsaturated oleic acid. Microbial community analysis reveals the ALK-2-CSTRs stimulated the growth of lipolytic bacteria and hydrogenotrophic methanogens, which suggests the hydrogenotrophic methanogenic pathway was promoted. Cost evaluation demonstrates the economical superiority of the ALK-2-CSTR over the prevailing strategies developed for enhancing methane yield from the co-substrates

Effect of Sodium on Methanogens in a Two-Stage Anaerobic System

This study evaluated the effects of sodium on anaerobic biomass from the second-stage reactor of a two-stage anaerobic digester. The results indicated that methanogens showed a relatively high sodium tolerance of 2.4 g Na+ L−1. Microbial community analysis showed that viable Methanomicrobiales was the most abundant population by a combined propidium monoazide cross-linking quantitative polymerase chain reaction technique. There was a population shift towards higher abundance of Thermotoga (0.02%), Clostridium (2.50%) and Methanoculleus (13.80%). Biomass activity in relation to increased sodium concentrations was investigated with the adenosine triphosphate test coupled with extracellular polymeric substances measurement. The results showed biomass activity decreased from 33 to 16 µg g−1 volatile suspended solids as sodium concentrations increased from 1.3 to 9.1 g Na+ L−1. Higher EPS production, particularly a greater predominance of carbohydrates, was stimulated by higher sodium concentrations. This study provides insights into the superiority of sodium tolerance of two-stage anaerobic digester in compared with a single-stage anaerobic system

The effect of pH on solubilization of organic matter and microbial community structures in sludge fermentation

Sludge fermentation between pH 4 and 11 was investigated to generate volatile fatty acids (VFA). Despite the highest sludge solubilization of 25.9% at pH 11, VFA accumulation was optimized at pH 8 (12.5% out of 13.1% sludge solubilization). 454 pyrosequencing identified wide diversity of acidogens in bioreactors operated at the various pHs, with Tissierella, Petrimonas, Proteiniphilum, Levilinea, Proteiniborus and Sedimentibacter enriched and contributing to the enhanced fermentation at pH 8. Hydrolytic enzymatic assays determined abiotic effect to be the leading cause for improved solubilization under high alkaline condition but the environmental stress at pH 9 and above might lead to disrupt biological activities and eventually VFA production. Furthermore, molecular weight (MW) characterization of the soluble fractions found large MW aromatic substances at pH 9 and above, that is normally associated with poor biodegradability, making them disadvantageous for subsequent bioprocesses. The findings provided information to better understand and control sludge fermentation.NRF (Natl Research Foundation, S’pore)Accepted versio

Impact of undissociated volatile fatty acids on acidogenesis in a two-phase anaerobic system

This study investigated the degradation and production of volatile fatty acids (VFAs) in the acidogenic phase reactor of a two-phase anaerobic system. 20 mmol/L bromoethanesulfonic acid (BESA) was used to inhibit acidogenic methanogens (which were present in the acidogenic phase reactor) from degrading VFAs. The impact of undissociated volatile fatty acids (unVFAs) on “net” VFAs production in the acidogenic phase reactor was then evaluated, with the exclusion of concurrent VFAs degradation. “Net” VFAs production from glucose degradation was partially inhibited at high unVFAs concentrations, with 59%, 37% and 60% reduction in production rates at 2190 mg chemical oxygen demand (COD)/L undissociated acetic acid (unHAc), 2130 mg COD/L undissociated propionic acid (unHPr) and 2280 mg COD/L undissociated n-butyric acid (unHBu), respectively. The profile of VFAs produced further indicated that while an unVFA can primarily affect its own formation, there were also unVFAs that affected the formation of other VFAs.NRF (Natl Research Foundation, S’pore

Effect of sodium on methanogens in a two-stage anaerobic system

This study evaluated the effects of sodium on anaerobic biomass from the second-stage reactor of a two-stage anaerobic digester. The results indicated that methanogens showed a rela-tively high sodium tolerance of 2.4 g Na+ L−1 . Microbial community analysis showed that viable Methanomicrobiales was the most abundant population by a combined propidium monoazide cross-linking quantitative polymerase chain reaction technique. There was a population shift towards higher abundance of Thermotoga (0.02%), Clostridium (2.50%) and Methanoculleus (13.80%). Biomass activity in relation to increased sodium concentrations was investigated with the adenosine triphos-phate test coupled with extracellular polymeric substances measurement. The results showed biomass activity decreased from 33 to 16 µg g−1 volatile suspended solids as sodium concentrations increased from 1.3 to 9.1 g Na+ L−1 . Higher EPS production, particularly a greater predominance of carbo-hydrates, was stimulated by higher sodium concentrations. This study provides insights into the superiority of sodium tolerance of two-stage anaerobic digester in compared with a single-stage anaerobic system.National Research Foundation (NRF)Published versionThe authors would like to thank the National Natural Science Foundation of China (No. 52170133). The authors would also like to thank the Singapore National Research Foundation for providing funding for the project “Wastewater Treatment Plants as Urban Eco Power Stations”

Comparison of single-stage and two-phase anaerobic sludge digestion systems : performance and microbial community dynamics

This study compared reactor performance and the respective microbial community dynamics in the conventional single-stage and 2-phase anaerobic digestion (AD) systems, treating municipal sludge to generate methane. The 2-phase system’s COD and VS reduction, and methane production could be maintained throughout the three HRTs tested (p = 0.05), which was associated with an increase in organic loading (30 d = 1.5 g COD L−1 d−1, 20 d = 2.2 g COD L−1 d−1 and 10 d = 3.5 g COD L−1 d−1); but this was not so in the single-stage system where it deteriorated at HRT of 10 d (p = 0.05) due to impairment of particulate COD reduction. qPCR, DGGE and the subsequent phylogenetic analysis revealed that microbial adaptation occurred as the seed sludge formed a different community in each reactor at 30 d HRT; however, no further significant microbial shift occurred at lower HRTs. The presence of specific hydrolytic and acidogenic Flavobacteriales and Clostriales in the acidogenic reactor may have allowed for enhanced hydrolysis and acidogenesis, leading to higher organic loading tolerance at 10 d HRT. Methanogenic activity in the acidogenic reactor may have been performed by Methanobacteriales and Methanosarcinaceae. Operation of the acidogenic reactor at neutral pH may have to be considered to ensure the cultivation of propionate oxidising bacteria, which could in turn, prevent reactor “souring” during high load conditions.NRF (Natl Research Foundation, S’pore)Accepted versio

Acetic acid effects on methanogens in the second stage of a two-stage anaerobic system

This study reports on biomass tolerance towards high concentrations of acetic acid (HAc) within the system. Biomass from the second stage of a two-stage anaerobic sludge digestion system was used for this study. Microbial community analysis by 454 pyrosequencing highlighted hydrogenotrophic Methanomicrobiales was the predominant archaeal population in the second stage (>99% of the total archaeal community). Second stage biomass degraded HAc up to 4200 mg HAc L-1 without observable lag phase. However, at HAc-shock loading of 7400 mg HAc L-1, it showed a one day lag phase associated with decreased biomass activity. After stepwise HAc-acclimation over 27 d, the biomass degraded HAc of up to 8200 mg HAc L-1 without observable lag phase. The dominance of Methanomicrobiales had remained unchanged in proportion - while the total archaeal population increased during acclimation. This study showed stepwise acclimation could be an approach to accommodate HAc accumulation and hence higher concentrations resulting from an enhanced first stage. Keywords: Acclimation; Acetic acid inhibition; Biomass activity; Hydrogenotrophic methanogens; Pyrosequencing; Two-stage anaerobic systemNRF (Natl Research Foundation, S’pore)Accepted versio