Pre-treatment with Trichoderma viride: Towards a better understanding of its consequences for anaerobic digestion.

Understanding and optimising biological pre-treatment strategies for enhanced bio-methane production is a central aspect in second-generation biofuel research. In this regard, the application of fungi for pre-treatment seems highly promising; however, understanding the mode of action is crucial. Here, we show how aerobic pre-treatment of crystalline cellulose with the cellulolytic Trichoderma viride affects substrate degradability during mesophilic, anaerobic digestion. It could be demonstrated that fungal pre-treatment resulted in a slightly reduced substrate mass. Nevertheless, no significant impact on the overall methane yield was found during batch fermentation. Short chain organic acids accumulation, thus, overall degradation dynamics including methane production kinetics were affected by the pre-treatment as shown by Gompertz modelling. Finally, 16S rRNA amplicon sequencing followed by ANCOM-BC resulted in up to 53 operative taxonomic units including fermentative, syntrophic and methanogenic taxa, whereby their relative abundances were significantly affected by fungal pre-treatment depending on the duration of the pre-treatment. The results demonstrated the impact of soft rot fungal pre-treatment of cellulose on subsequent anaerobic cellulose hydrolysis as well as on methanogenic activity. To the best of our knowledge, this is the first study to investigate the direct causal effects of pre-treatment with T. viride on basic but crucial anaerobic digestion parameters in a highly standardised approach.

Gasification chars and activated carbon: Systematic physico-chemical characterization and effect on biogas production.

Gasification residues/chars (GR) and activated carbon (AC) are added to wastewater treatment processes mainly as a fourth purification stage, e.g., to adsorb heavy metals or pharmaceutical residues. However, the effects of GR or AC, which are transferred to the anaerobic digestion (AD) via the sludge, are not yet fully understood. Although, the positive effect of char addition on AD has been demonstrated in several investigations, systematic studies with chemically well described chars are still missing. Therefore, in this study, different chars were characterized in detail, subjected to AD in different concentrations, and their effect on methane production investigated. GR of a gasification plant with a floating fixed bed technology, carbon made by chemical impregnation with ZnCl2 from waste-wood, carbon produced by thermochemical activation with CO2 from GR and commercial powdered AC were used for the experiments. Among others, thermogravimetric analysis, physisorption, pH, and conductivity analysis were used to characterize the chars. Mesophilic AD batch tests with different concentrations (0.025, 0.05, 0.5, 1.0, 7.0, 14.0 gL-1) of all chars (GR and ACs, respectively) were performed with digester sludge from a wastewater treatment plant for a period of 47 d. Volatile fatty acids (VFA) as well as biogas production and CH4 concentrations were monitored. It could be shown, that concentrations below 1.0 g char L-1 did not result in significant effects on CH4 and/or VFA production, whereas high concentrations of GR and AC influenced both, the CH4 yield and kinetics. Depending on the production process and the characteristics of the chars, the effect on AD varied, whereby both, positive and negative effects on biogas yield and methane production were observed. This study provides the first systematic evaluation of char application to AD processes, and therefore allows for better predictions of char applicability and effect.

Meso- and thermophilic posttreatment of press water coming from a thermophilic municipal solid waste digester.

An efficient biogas production out of organic (waste) materials is important to contribute to a carbon-neutral future. In this study, thermophilic press water (PW) coming from an organic fraction of the municipal solid waste digester was further digested in a thermo- and mesophilic posttreatment approach using two semicontinuous 14 L digesters. The results showed that the PW can still have considerable high biogas potential-at least during the touristic high season in central Europe. The change in temperature led to an increase in volatile fatty acid concentrations and a decrease in biogas production in the mesophilic approach in the first days. However, the losses in biogas production at the beginning could be compensated thus there were no considerable differences in biogas production between thermo- and mesophilic posttreatment at the end of incubation. This can most probably be contributed to a change in the microbial community, and potentially problematic intermediates like valerate could be better degraded in the mesophilic reactor. Especially the abundance of representatives of the phylum Bacteroidota, like Fermentimonas spp., increased during mesophilic anaerobic digestion.

Effects of phenyl acids on different degradation phases during thermophilic anaerobic digestion.

Aromatic compounds like phenyl acids (PA) can accumulate during anaerobic digestion (AD) of organic wastes due to an increased entry of lignocellulose, secondary plant metabolites or proteins, and thermodynamic challenges in degrading the benzene ring. The effects of aromatic compounds can be various - from being highly toxic to be stimulating for methanogenesis - depending on many parameters like inoculum or molecular characteristics of the aromatic compound. To contribute to a better understanding of the consequences of PA exposure during AD, the aim was to evaluate the effects of 10 mM PA on microbial communities degrading different, degradation phase-specific substrates in thermophilic batch reactors within 28 days: Microcrystalline cellulose (MCC, promoting hydrolytic to methanogenic microorganisms), butyrate or propionate (promoting syntrophic volatile fatty acid (VFA) oxidisers to methanogens), or acetate (promoting syntrophic acetate oxidisers to methanogens). Methane production, VFA concentrations and pH were evaluated, and microbial communities and extracellular polymeric substances (EPS) were assessed. The toxicity of PA depended on the type of substrate which in turn determined the (i) microbial diversity and composition and (ii) EPS quantity and quality. Compared with the respective controls, methane production in MCC reactors was less impaired by PA than in butyrate, propionate and acetate reactors which showed reductions in methane production of up to 93%. In contrast to the controls, acetate concentrations were high in all PA reactors at the end of incubation thus acetate was a bottle-neck intermediate in those reactors. Considerable differences in EPS quantity and quality could be found among substrates but not among PA variants of each substrate. Methanosarcina spp. was the dominant methanogen in VFA reactors without PA exposure and was inhibited when PA were present. VFA oxidisers and Methanothermobacter spp. were abundant in VFA assays with PA exposure as well as in all MCC reactors. As MCC assays showed higher methane yields, a higher microbial diversity and a higher EPS quantity and quality than VFA reactors when exposed to PA, we conclude that EPS in MCC reactors might have been beneficial for absorbing/neutralising phenyl acids and keeping (more susceptible) microorganisms shielded in granules or biofilms.

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples.

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.

Lignin intermediates lead to phenyl acid formation and microbial community shifts in meso- and thermophilic batch reactors.

BACKGROUND: Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. RESULTS: The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. CONCLUSIONS: PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.

Microbial community dynamics in mesophilic and thermophilic batch reactors under methanogenic, phenyl acid-forming conditions.

BACKGROUND: Proteinaceous wastes exhibit high theoretical methane yields and their residues are considered valuable fertilisers. The routine anaerobic degradation of proteins often raises problems like high aromatic compound concentrations caused by the entry of aromatic amino acids into the system. A profound investigation of the consequences of aromatic compound exposure on various microorganisms, which cascade-like and interdependently degrade complex molecules to biogas, is still pending. RESULTS: In mesophilic samples, methane was predominantly produced via acetoclastic methanogenesis. The highest positive correlation was observed between phenylacetate (PAA) and Psychrobacter spp. and between phenylpropionate (PPA) and Haloimpatiens spp. Moreover, Syntrophus spp. negatively correlated with PAA (Spearman's rank correlations coefficient (rs) = - 0.46, p < 0.05) and PPA concentrations (rs = - 0.44, p < 0.05) and was also associated with anaerobic benzene ring cleavage. In thermophilic samples, acetate was predominantly oxidised by Tepidanaerobacter spp. or Syntrophaceticus spp. in syntrophic association with a hydrogenotrophic methanogen. The genera Sedimentibacter and Syntrophaceticus correlated positively with both PAA and PPA concentrations. Moreover, Sedimentibacter spp., Tepidanaerobacter spp., Acetomicrobium spp., and Sporanaerobacter spp. were significant LEfSe (linear discriminant analysis effect size) biomarkers for high meso- as well as thermophilic phenyl acid concentrations. Direct negative effects of phenyl acids on methanogenic properties could not be proven. CONCLUSIONS: Anaerobic phenyl acid formation is not restricted to specific microbial taxa, but rather done by various meso- and thermophilic bacteria. The cleavage of the highly inert benzene ring is possible in methanogenic batch reactors-at least in mesophilic fermentation processes. The results indicated that phenyl acids rather affect microorganisms engaged in preceding degradation steps than the ones involved in methanogenesis.

Microbial and Phenyl Acid Dynamics during the Start-up Phase of Anaerobic Straw Degradation in Meso- and Thermophilic Batch Reactors.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation.

Formation of phenylacetic acid and phenylpropionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion.

BACKGROUND: Substrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions. METHODS: Different states of overload were simulated in batch experiments while reviewing the generation of phenyl acids out of different lab-use substrates in order to evaluate the impact on biogas and methane production as well as some additional process performance parameters under defined laboratory conditions. Investigations were conducted under both mesophilic and thermophilic conditions. RESULTS: It could be shown that the tested input materials led to the formation of phenyl acids in a substrate-dependent manner with the formation itself being less temperature driven. Once formed, the formation of phenyl acids turned out to be a reversible process. CONCLUSIONS: Although a mandatory negative impact of phenyl acids per se on the anaerobic digestion process in general and the methanogenesis process in particular could not be proven, phenyl acids, however, seem to play an important role in the microbial response to overloaded biogas systems.

Biological Pretreatment Strategies for Second-Generation Lignocellulosic Resources to Enhance Biogas Production.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.