Microbiological Surveillance of Biogas Plants: Targeting Acetogenic Community

Acetogens play a very important role in anaerobic digestion and are essential in ensuring process stability. Despite this, targeted studies of the acetogenic community in biogas processes remain limited. Some efforts have been made to identify and understand this community, but the lack of a reliable molecular analysis strategy makes the detection of acetogenic bacteria tedious. Recent studies suggest that screening of bacterial genetic material for formyltetrahydrofolate synthetase (FTHFS), a key marker enzyme in the Wood-Ljungdahl pathway, can give a strong indication of the presence of putative acetogens in biogas environments. In this study, we applied an acetogen-targeted analyses strategy developed previously by our research group for microbiological surveillance of commercial biogas plants. The surveillance comprised high-throughput sequencing of FTHFS gene amplicons and unsupervised data analysis with the AcetoScan pipeline. The results showed differences in the acetogenic community structure related to feed substrate and operating parameters. They also indicated that our surveillance method can be helpful in the detection of community changes before observed changes in physico-chemical profiles, and that frequent high-throughput surveillance can assist in management towards stable process operation, thus improving the economic viability of biogas plants. To our knowledge, this is the first study to apply a high-throughput microbiological surveillance approach to visualise the potential acetogenic population in commercial biogas digesters

Acetate and Lactate Production During Two-Stage Anaerobic Digestion of Food Waste Driven by Lactobacillus and Aeriscardovia

Background: In a previous study, single-stage processes were compared with two-stage processes, using either food waste alone or mixed with thin stillage as substrate. Overall methane yield increased (by 12%) in two-stage compared with single-stage digestion when using food waste, but decreased when food waste was co-digested with thin stillage (50:50 on VS basis). The obtained difference in methane yield was likely caused by a higher acetate level in the first stage reactor operating with food waste alone (around 20 g/L) compared to the reactor also treating thin stillage (around 8 g /L). The present study sought to shed additional light on possible causes of the large difference in methane yield by scrutinizing the microbial community in the first- and second-stage reactors, using a combined Illumina sequencing and qPCR approach. Results: In the first-stage process, acid-tolerant Aeriscardovia and Lactobacillus formed a highly efficient consortium. For food waste with high levels of acetate (20 g/L, equal to 0.14 g acetate/g VS) was produced but when thin stillage was added the pH was lower (<4), resulting in lactate production exceeding acetate production. This difference in hydrolysate composition between the reactors resulted in development of slightly different communities in the second-stage, for both hydrolysis, fermentation, and acetogenesis. High acetate concentration appeared to promote proliferation of different syntrophic consortia, such as various syntrophic acetate oxidizers, members of the genus Syntrophomonas and candidate phylum Cloacimonetes, likely explaining the higher methane yields with two-step compared with single-stage digestion of food waste.Conclusion: Using food waste as sole substrate resulted in enrichment of Lactobacillus and Aeriscardovia and high acetate yields in the first-stage reactor. This was beneficial for biogas yield in two-stage digestion, where efficient acid-degrading syntrophic consortia developed. Addition of thin stillage contributed to low pH and higher lactate production, which resulted in decreased methane yield in the two-stage process compared with using food waste as sole substrate

Serial anaerobic digestion improves protein degradation and biogas production from mixed food waste

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps

Biogas production through syntrophic acetate oxidation and deliberate operating strategies for improved digester performance

Anaerobic degradation of protein-rich materials has high methane potential and produces nutrient-rich residue, but requires strategies to avoid ammonia inhibition. A well-adapted process can cope with substantially higher ammonia levels than an unadapted process and analyses of pathways for methanisation of acetate, combined with determination of microbial community structure, strongly indicate that this is due to a significant contribution of syntrophic acetate oxidation. The microorganisms involved in syntrophic acetate oxidation thus most likely occupy a unique niche and play an important role in methane formation. This review summarises current insight of syntrophic acetate oxidising microorganisms, their presence and the detection of novel species and relate these observations with operating conditions of the biogas processes in order to explore contributing factors for development of an ammonia-tolerant microbial community that efficiently degrades acetate through the syntrophic pathway. Besides high ammonia level, acetate concentration, temperature and methanogenic community structure are considered in this review as likely factors that shape and influence SAO-mediated microbial ecosystems. The main purpose of this review is to facilitate process optimisation through considering the activity and growth of this key microbial community

Identifying targets for increased biogas production through chemical and organic matter characterization of digestate from full-scale biogas plants: what remains and why?

Background This study examines the destiny of macromolecules in different full-scale biogas processes. From previous studies it is clear that the residual organic matter in outgoing digestates can have significant biogas potential, but the factors dictating the size and composition of this residual fraction and how they correlate with the residual methane potential (RMP) are not fully understood. The aim of this study was to generate additional knowledge of the composition of residual digestate fractions and to understand how they correlate with various operational and chemical parameters. The organic composition of both the substrates and digestates from nine biogas plants operating on food waste, sewage sludge, or agricultural waste was characterized and the residual organic fractions were linked to substrate type, trace metal content, ammonia concentration, operational parameters, RMP, and enzyme activity. Results Carbohydrates represented the largest fraction of the total VS (32-68%) in most substrates. However, in the digestates protein was instead the most abundant residual macromolecule in almost all plants (3-21 g/kg). The degradation efficiency of proteins generally lower (28-79%) compared to carbohydrates (67-94%) and fats (86-91%). High residual protein content was coupled to recalcitrant protein fractions and microbial biomass, either from the substrate or formed in the degradation process. Co-digesting sewage sludge with fat increased the protein degradation efficiency with 18%, possibly through a priming mechanism where addition of easily degradable substrates also triggers the degradation of more complex fractions. In this study, high residual methane production (> 140 L CH4/kg VS) was firstly coupled to operation at unstable process conditions caused mainly by ammonia inhibition (0.74 mg NH3-N/kg) and/or trace element deficiency and, secondly, to short hydraulic retention time (HRT) (55 days) relative to the slow digestion of agricultural waste and manure. Conclusions Operation at unstable conditions was one reason for the high residual macromolecule content and high RMP. The outgoing protein content was relatively high in all digesters and improving the degradation of proteins represents one important way to increase the VS reduction and methane production in biogas plants. Post-treatment or post-digestion of digestates, targeting microbial biomass or recalcitrant protein fractions, is a potential way to achieve increased protein degradation

Biogas production from thin stillage

The biogas plant in Norrköping (Tekniska verken i Linköping AB, publ.), Sweden, operates with thin stillage, a residue from bio-ethanol fermentation, as the main feedstock. Thin stillage is energy-rich due to its high protein content, but due to its high nitrogen and sulphate content is a somewhat complicated feedstock. The high nitrogen concentration results in inhibition of the microbial process and also selects for nitrogen-tolerant, but slow-growing, syntrophic acetate-oxidising bacteria (SAOB). The high sulphate concentration in the feedstock results in production of toxic and inhibitory sulphides through the activity of sulphate-reducing bacteria (SRB). Measures currently applied at Norrköping biogas plant to optimise the degradation of thin stillage include: i) use of mesophilic temperature and addition of hydrochloric acid, ii) use of long hydraulic retention time and iii) addition of iron and trace elements. This thesis investigated how to obtain a more efficient biogas process treating thin stillage, with Norrköping biogas plant as the model plant. It also explored the role of SRB in the anaerobic process at high nitrogen content and sought to identify optimal conditions for ammonia-tolerant methane-producing microorganisms. This was done by measuring SRB abundance in several large-scale biogas processes to identify conditions resulting in reduced numbers. In parallel, the effects of increasing temperature and organic load, calcium addition and a two-stage strategy were evaluated in laboratory studies. The results showed a correlation between high ammonia level and temperature with decreased abundance of SRB, but none of the operating strategies tested proved successful in repressing sulphate reduction. However, increasing ammonia and/or organic loading rate influenced both the acetogenic and methanogenic community, including potential SAOB. Moreover, increasing the temperature to 44 ºC resulted in increased abundance of thermotolerant SAOB and their partner methanogen and higher biogas yield (+22%). A maximum ammonia threshold concentration of approximately 1.1 g L-1 was identified. Application of the findings reported in this thesis has resulted in increased process stability in biogas plants in Sweden

Evaluation of magnetic biomass carriers for biogas production

This thesis evaluates a novel technique to increase the active biomass inside continuously stirred tank biogas reactors with possible benefits of shorter retention times, higher degree of degradation, higher methane yield and tolerance of higher organic loading rates. The technique includes addition of magnetic biomass carriers to the process which, after adhesion of active microorganisms, can be magnetically separated at reactor outflow and reintroduced to the process. The evaluation of magnetic biomass carriers included methods such as batch experiments, quantitative real-time polymerase chain reaction and continuous reactor experiments with different organic loading rates and addition of volatile fatty acids. The results show that reintroduction of magnetic biomass carriers does indeed work: an accumulated biomass of microorganisms is achieved inside the reactor during a continuous process. Magnetite was selected as the most promising biomass carrier, microbiological studies of the particles show that microbiological colonization of magnetite is present with preferential adhesion of hydrogenotrophic methanogens, important for the methanogenesis. The anaerobic digestion with magnetite as biomass carrier present increased process stability and elevated degrading potential of volatile fatty acids, as well as leading to higher methane content when subjected to increased organic load. Thus, the total gas production is increased in certain situations when using magnetic biomass carriers, why further studies of appropriate hydraulic retention times, organic loading rates and substrates are warranted

Inoculum source determines acetate and lactate production during anaerobic digestion of sewage sludge and food waste

Acetate production from food waste or sewage sludge was evaluated in four semi-continuous anaerobic digestion processes. To examine the importance of inoculum and substrate for acid production, two different inoculum sources (a wastewater treatment plant (WWTP) and a co-digestion plant treating food and industry waste) and two common substrates (sewage sludge and food waste) were used in process operations. The processes were evaluated with regard to the efficiency of hydrolysis, acidogenesis, acetogenesis, and methanogenesis and the microbial community structure was determined. Feeding sewage sludge led to mixed acid fermentation and low total acid yield, whereas feeding food waste resulted in the production of high acetate and lactate yields. Inoculum from WWTP with sewage sludge substrate resulted in maintained methane production, despite a low hydraulic retention time. For food waste, the process using inoculum from WWTP produced high levels of lactate (30 g/L) and acetate (10 g/L), while the process initiated with inoculum from the co-digestion plant had higher acetate (25 g/L) and lower lactate (15 g/L) levels. The microbial communities developed during acid production consisted of the major genera Lactobacillus (92–100%) with food waste substrate, and Roseburia (44–45%) and Fastidiosipila (16–36%) with sewage sludge substrate. Use of the outgoing material (hydrolysates) in a biogas production system resulted in a non-significant increase in bio-methane production (+5–20%) compared with direct biogas production from food waste and sewage sludge

Determination of Methane and Carbon Dioxide Formation Rate Constants for Semi-Continuously Fed Anaerobic Digesters

To optimize commercial-scale biogas production, it is important to evaluate the performance of each microbial step in the anaerobic process. Hydrolysis and methanogenesis are usually the rate-limiting steps during digestion of organic waste and by-products. By measuring biogas production and methane concentrations on-line in a semi-continuously fed reactor, gas kinetics can be evaluated. In this study, the rate constants of the fermentative hydrolysis step (kc) and the methanogenesis step (km) were determined and evaluated in a continuously stirred tank laboratory-scale reactor treating food and slaughterhouse waste and glycerin. A process additive containing Fe2+, Co2+ and Ni2+ was supplied until day 89, after which Ni2+ was omitted. The omission resulted in a rapid decline in the methanogenesis rate constant (km) to 70% of the level observed when Ni2+ was present, while kc remained unaffected. This suggests that Ni2+ mainly affects the methanogenic rather than the hydrolytic microorganisms in the system. However, no effect was initially observed when using conventional process monitoring parameters such as biogas yield and volatile fatty acid concentration. Hence, formation rate constants can reveal additional information on process performance and km can be used as a complement to conventional process monitoring tools for semi-continuously fed anaerobic digesters

Post-treatment of dewatered digested sewage sludge by thermophilic high-solid digestion for pasteurization with positive energy output

This study investigated the possibility to use thermophilic anaerobic high solid digestion of dewatered digested sewage sludge (DDS) at a wastewater treatment plant (WWTP) as a measure to increase total methane yield, achieve pasteurization and reduce risk for methane emissions during storage of the digestate. A pilot-scale plug-flow reactor was used to mimic thermophilic post-treatment of DDS from a WWTP in Linkoping, Sweden. Process operation was evaluated with respect to biogas process performance, using both chemical and microbiological parameters. Initially, the process showed disturbance, with low methane yields and high volatile fatty acid (VFA) accumulation. However, after initiation of digestate recirculation performance improved and the specific methane production reached 46 mL CH4/g VS. Plug flow conditions were assessed with lithium chloride and the hydraulic retention time (HRT) was determined to be 19-29 days, sufficient to reach successful pasteurization. Degradation rate of raw protein was high and resulted in ammonia-nitrogen levels of up to 2.0 g/L and a 30% lower protein content in the digestate as compared to DDS. Microbial analysis suggested a shift in the methane producing pathway, with dominance of syntrophic acetate oxidation and the candidate methanogen family WSA2 by the end of the experiment. Energy balance calculations based on annual DDS production of 10 000 ton/year showed that introduction of high-solid digestion as a post-treatment and pasteurization method would result in a positive energy output of 340 MWh/year. Post-digestion of DDS also decreased residual methane potential (RMP) by >96% compared with fresh DDS. (C) 2020 The Authors. Published by Elsevier Ltd