Enhancing biogas production from lignocellulosic digestate through priming and post-digestion

The application of priming, the increase of microbial organic matter turnover by the addition of easily degradable carbon, was investigated for improving biogas production and post-digestion efficiency. Continuously stirred tank main- and post-digesters were operated on manure-based agricultural waste. Potato starch was added as the priming substrate to the post-digester. Addition of 0.2 g VS/L starch every other day increased biogas production from 413 f 97 to 509 f 58 mL biogas/day. The increased biogas production exceeds the output from the individual substrates confirming the priming effect. Additionally, the specific biomethane production increased from 74 f 16 to 116 f 43 mL/g VS. A shift in microbial community was detected, benefiting lignocellulolytic activities, as degradation of cellulose, hemicellulose and total sugar was enhanced. The results indicate successful application of priming which can improve the overall efficiency of biogas production from manure-based feedstock

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

Impact of additives on syntrophic propionate and acetate enrichments under high-ammonia conditions

High ammonia concentrations in anaerobic degradation systems cause volatile fatty acid accumulation and reduced methane yield, which often derive from restricted activity of syntrophic acid-oxidising bacteria and hydrogenotrophic methanogens. Inclusion of additives that facilitate the electron transfer or increase cell proximity of syntrophic species by flocculation can be a suitable strategy to counteract these problems, but its actual impact on syntrophic interactions has yet to be determined. In this study, microbial cultivation and molecular and microscopic analysis were performed to evaluate the impact of conductive (graphene, iron oxide) and non-conductive (zeolite) additives on the degradation rate of acetate and propionate to methane by highly enriched ammonia-tolerant syntrophic cultures derived from a biogas process. All additives had a low impact on the lag phase but resulted in a higher rate of acetate (except graphene) and propionate degradation. The syntrophic bacteria 'Candidatus Syntrophopropionicum ammoniitolerans', Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen were found in higher relative abundance and higher gene copy numbers in flocculating communities than in planktonic communities in the cultures, indicating benefits to syntrophs of living in close proximity to their cooperating partner. Microscopy and element analysis showed precipitation of phosphates and biofilm formation in all batches except on the graphene batches, possibly enhancing the rate of acetate and propionate degradation. Overall, the concordance of responses observed in both acetate- and propionate-fed cultures highlight the suitability of the addition of iron oxide or zeolites to enhance acid conversion to methane in high-ammonia biogas processes

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

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

AcetoBase: a functional gene repository and database for formyltetrahydrofolate synthetase sequences

Acetogenic bacteria are imperative to environmental carbon cycling and diverse biotechnological applications, but their extensive physiological and taxonomical diversity is an impediment to systematic taxonomic studies. Acetogens are chemolithoautotrophic bacteria that perform reductive carbon fixation under anaerobic conditions through the Wood–Ljungdahl pathway (WLP)/acetyl-coenzyme A pathway. The gene-encoding formyltetrahydrofolate synthetase (FTHFS), a key enzyme of this pathway, is highly conserved and can be used as a molecular marker to probe acetogenic communities. However, there is a lack of systematic collection of FTHFS sequence data at nucleotide and protein levels. In an attempt to streamline investigations on acetogens, we developed AcetoBase - a repository and database for systematically collecting and organizing information related to FTHFS sequences. AcetoBase also provides an opportunity to submit data and obtain accession numbers, perform homology searches for sequence identification and access a customized blast database of submitted sequences. AcetoBase provides the prospect to identify potential acetogenic bacteria, based on metadata information related to genome content and the WLP, supplemented with FTHFS sequence accessions, and can be an important tool in the study of acetogenic communities. AcetoBase can be publicly accessed at https://acetobase.molbio.slu.se

Evaluating the effect of biochar addition on the anaerobic digestion of swine manure: application of Py-GC/MS

The anaerobic digestion process of swine manure was studied when char was used as supplement for improving performance. The use of pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) was proposed for assessing the organic matter degradation. The assessment on biogas production was carried out using samples of swine manure (SM) supplemented with char in one case and pre-treated by microwave irradiation in the other. This experimental set-up allows for the comparison of the biological degradation observed under these two different configurations and therefore aids in understanding the effect of char particles on the process. Results showed similar performance for both systems, with an average improvement of 39% being obtained in methane production when compared to the single digestion of SM. The analysis of digestate samples by Fourier transform infrared (FTIR) spectroscopy and Py-GC/MS showed improved degradation of proteins, with the Py-GC/MS technique also capable of identifying an increase in microbial-derived material when char was added, therefore highlighting the relevant role of carbon conductive particles on biological systems. Py-GC/MS along with the use of FTIR spectroscopy has proven to be useful tools when evaluating anaerobic digestion

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