The recalcitrant structures either from substrate or microbial biomass contained in digestates after anaerobic digestion (AD) highly influence digestate valorization. To properly assess the microbial biomass contribution to the digested or-ganic matter (OM), a combination of characterization methods and the use of various substrate types in anaerobic con-tinuous reactors was required. The use of totally biodegradable substrates allowed detecting soluble microbial products via fluorescence spectroscopy at emission wavelengths of 420 and 460 nm while the protein-like signature was enhanced by the whey protein. During reactors operation, a transfer of complex compounds to the dissolved OM from the particulate OM was observed through fluorescence applied on biochemical fractionation. Consequently, the fluorescence complexity index of the dissolved OM increased from 0.59-0.60 to 1.06-1.07, whereas it decreased inversely for the extractable soluble from the particulate OM from 1.16-1.19 to 0.42-0.54. Accordingly, fluorescence regional integration showed differences among reactors based on visual inspection and orthogonal partial latent struc-tures (OPLS) analysis. Similarly, the impact of the substrate type and operation time on the particulate OM was revealed by 13C nuclear magnetic resonance using OPLS, providing a good model (R2X = 0.93 and Q2 = 0.8) with a clear time-trend. A high signal resonated at similar to 30 ppm attributed to CH2-groups in the aliphatic chain of lipid-like struc-ture besides carbohydrates intensities at 60-110 ppm distinguished the reactor fed with whey protein from the other, which was mostly biomass related. Indeed, this latter displayed a higher presence of peptidoglycan (delta H/C: 1.6-2.0/ 20-25 ppm) derived from microbial biomass by 1H -13C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance. Interestingly, the sample distribution obtained by non-metric multidimensional scaling of bacte-rial communities resembled the attained using 13C NMR properties, opening new research perspectives. Overall, this study discloses the microbial biomass contribution to digestates composition to improve the OM transformation mech-anism knowledge.

Anaerobic digestion (AD) is an important technology for achieving sustainability, but it faces challenges in meeting rising production demands while remaining economically profitable. One difficulty is the lack of a comprehensive understanding of the many interactions within anaerobic digesters, which makes it challenging to fully optimize them. This is particularly notable when considering the interlinked dynamics between micronutrient availability and fluid behavior. This study addresses this gap by focusing on key operational parameters affecting the efficiency of the process in continuous stirred-tank biogas reactors, which are the most used AD technology today. It does so by proposing and evaluating a novel conceptual model of the mechanisms behind how different parts of AD processes interact upon disturbance, highlighting strategies for preventing process failure. This article aims to improve our understanding of the complexity of AD biotechnology and to provide a starting point for developing advanced strategies for operational optimization.

Sulfide ions are regarded to be toxic to microorganisms in engineered methanogenic systems (EMS), where organic substances are anaerobically converted to products such as methane, hydrogen, alcohols, and carboxylic acids. A vast body of research has addressed solutions to mitigate process disturbances associated with high sulfide levels, yet the established paradigm has drawn the attention away from the multifaceted sulfide interactions with minerals, organics, microbial interfaces and their implications for performance of EMS. This brief review brings forward sulfide-derived pathways other than toxicity and with potential significance for anaerobic organic matter degradation. Available evidence on sulfide reactions with organic matter, interventions with key microbial metabolisms, and interspecies electron transfer are critically synthesized as a guidance for comprehending the sulfide effects on EMS apart from the microbial toxicity. The outcomes identify existing knowledge gaps and specify future research needs as a step forward towards realizing the potential of sulfide-derived mechanisms in diversifying and optimizing EMS applications.

Natural environments with frequent drainage experience drying and rewetting events that impose fluctuations in water availability and oxygen exposure. These relatively dramatic cycles profoundly impact microbial activity in the environment and subsequent emissions of methane and carbon dioxide. In this study, we mimicked drying and rewetting events by submitting methanogenic communities from strictly anaerobic environments (anaerobic digestors) with different phylogenetic structures to consecutive desiccation events under aerobic (air) and anaerobic (nitrogen) conditions followed by rewetting. We showed that methane production quickly recovered after each rewetting, and surprisingly, no significant difference was observed between the effects of the aerobic or anaerobic desiccation events. There was a slight change in the microbial community structure and a decrease in methane production rates after consecutive drying and rewetting, which can be attributed to a depletion of the pool of available organic matter or the inhibition of the methanogenic communities. These observations indicate that in comparison to the drying and rewetting events or oxygen exposure, the initial phylogenetic structure and the organic matter quantity and quality exhibited a stronger influence on the methanogenic communities and overall microbial community responses. These results change the current paradigm of the sensitivity of strict anaerobic microorganisms to oxygen exposure.

This study aims to elucidate the role of sulfide and its precursors in anaerobic digestion (i.e., cysteine, representing sulfur-containing amino acids, and sulfate) on microbial oleate conversion to methane. Serine, with a similar structure to cysteine but with a hydroxyl group instead of a thiol, was included as a control to assess potential effects on methane formation that were not related to sulfur functionalities. The results showed that copresence of sulfide and oleate in anaerobic batch assays accelerated the methane formation compared to assays with only oleate and mitigated negative effect on methane formation caused by increased sulfide level. Nuclear magnetic resonance spectroscopy of sulfide exposed oleate suggested that sulfide reaction with oleate double bonds likely contributed to negation of the negative effect on the methanogenic activity. Methane formation from oleate was also accelerated in the presence of cysteine or serine, while sulfate decreased the cumulative methane formation from oleate. Neither cysteine nor serine was converted to methane, and their accelerating effects was associated to different mechanisms due to establishment of microbial communities with different structures, as evidenced by high-throughput sequencing of 16S rRNA gene. These outcomes contribute with new knowledge to develop strategies for optimum use of sulfur-and lipid-rich wastes in anaerobic digestion processes.

Biogas production by anaerobic digestion (AD) of organic wastes is important for the transition to fossil free fuelsin both the transport sector, industries and shipping. The aim of this study was to target the residual organicmatter in the outgoing residue from the AD process, so called digestate, with different thermal treatmentmethods in order to improve digestate degradability and biogas potential upon post-digestion. The thermaltreatment was performed at 55 ◦C in 24 h, 70 ◦C in 1 h and by thermal hydrolysis process (THP; 165 ◦C, 8 bar in0.33 h), and were carefully selected to offer a simultaneous possibility for pasteurization of the digestate accordingto the regulations in Sweden. Digestates from ten full-scale biogas plants were collected, with differentsubstrate profiles including wastewater treatment plant (WWTP), food waste digestion, agriculture digestion andmanure digestion. The results showed that all thermal treatment methods caused increased dissolved organiccarbon concentration (DOC). Four of the thermal treated digestates with the highest increase in DOC weresubsequently tested for the bio-methane potential. Thermal treatments at 70 ◦C and THP, respectively, resulted inthe highest increase in bio-methane potentials, with an increase of 15–39% for one WWTP, 38 – 40% fordigestate from an agriculture digestion plant and 20 – 22% for digestate from a co-digestion plant treating foodwaste. Interestingly, the bio-methane potential from digestate treated with the energy-intense THP method, didnot show any significant difference compared to thermal treatment at 70 ◦C for 1 h. The outcomes of this studysuggest that placing a pasteurization unit between a main digester and a post digester, when applying two-stepdigestion allows for a combined pasteurization and increased biogas production.

Background Slow degradation kinetics of long-chain fatty acids (LCFA) and their accumulation in anaerobic digesters disrupt methanogenic activity and biogas production at high loads of waste lipids. In this study, we evaluated the effect of effluent solids recirculation on microbial LCFA (oleate) degradation capacity in continuous stirred-tank sludge digesters, with the overall aim of providing operating conditions for efficient co-digestion of waste lipids. Furthermore, the impacts of LCFA feeding frequency and sulfide on process performance and microbial community dynamics were investigated, as parameters that were previously shown to be influential on LCFA conversion to biogas. Results Effluent solids recirculation to municipal sludge digesters enabled biogas production of up to 78% of the theoretical potential from 1.0 g oleate l(-1) day(-1). In digesters without effluent recirculation, comparable conversion efficiency could only be reached at oleate loading rates up to 0.5 g l(-1) day(-1). Pulse feeding of oleate (supplementation of 2.0 g oleate l(-1) every second day instead of 1.0 g oleate l(-1) every day) did not have a substantial impact on the degree of oleate conversion to biogas in the digesters that operated with effluent recirculation, while it marginally enhanced oleate conversion to biogas in the digesters without effluent recirculation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea revealed that pulse feeding resulted in prevalence of fatty acid-degrading Smithella when effluent recirculation was applied, whereas Candidatus Cloacimonas prevailed after pulse feeding of oleate in the digesters without effluent recirculation. Combined oleate pulse feeding and elevated sulfide level contributed to increased relative abundance of LCFA-degrading Syntrophomonas and enhanced conversion efficiency of oleate, but only in the digesters without effluent recirculation. Conclusions Effluent solids recirculation improves microbial LCFA degradation capacity, providing possibilities for co-digestion of larger amounts of waste lipids with municipal sludge.

Nanosized metal derivatives (NMDs), referring to metals and their oxides, are extensively utilized as additives for anaerobic digestion (AD) and dark fermentation (DF) processes, for enhancing the production of methane (CH4) and hydrogen (H-2), respectively. NMDs-derived positive impacts were widely confirmed in many previous studies; however, no consensus exists about how these have been acquired. Undoubtedly, NMDs affect extracellular electron transfer (EET). Consequently, we explore how biotic-biotic interactions, referring to direct interspecies electron transfer (DIET) among AD partners, and biotic-abiotic exchanges, which are mediated by redox reactions with metals, are affected. In this perspective, the mechanisms behind all those effects are reviewed and explained in detail, considering the specific properties of each NMD, e.g., size and type. We discuss previous studies that offer contradicting interpretations about which process dominates metal oxidation, metal reduction, or DIET. In addition, the fate of NMDs residues in the digestate after the treatment process is discussed, focusing on NMDs toxicity. From previous literature, the environmental impacts are evaluated for the production process of NMDs that are utilized in AD and DF processes via life-cycle assessment. This review provides a comprehensive understanding of NMDs-microbes interactions, which are mandatory for (i) building clear scientific knowledge about processes in play and (ii) engineering favorable conditions to achieve optimum yields in AD and DF processes.

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 Linköping, 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.

Trace elements (TEs) are vital for anaerobic digestion (AD), due to their role as cofactors in many key enzymes. The aim of this study was to evaluate the effects of specific TE deficiencies on mixed microbial communities during AD of soluble polymer-free substrates, thus focusing on AD after hydrolysis. Three mesophilic (37 degrees C) continuous stirred-tank biogas reactors were depleted either of Co, Ni, or a combination of Se and W, respectively, by discontinuing their supplementation. Ni and Se/W depletion led to changes in methane kinetics, linked to progressive volatile fatty acid (VFA) accumulation, eventually resulting in process failure. No significant changes occurred in the Co-depleted reactor, indicating that the amount of Co present in the substrate in absence of supplementation was sufficient to maintain process stability. Archaeal communities remained fairly stable independent of TE concentrations, while bacterial communities gradually changed with VFA accumulation in Ni- and Se-/W-depleted reactors. Despite this, the communities remained relatively similar between these two reactors, suggesting that the major shifts in composition likely occurred due to the accumulating VFAs. Overall, the results indicate that Ni and Se/W depletion primarily lead to slower metabolic activities of methanogenic archaea and their syntrophic partners, which then has a ripple effect throughout the microbial community due to a gradual accumulation of intermediate fermentation products.

During the process of pulp- and papermaking, large volumes of fibre-rich primary sludge are generated. Anaerobic digestion of primary sludge offers a substantial potential for methane production as an alternative approach to the inefficient energy recoveries by commonly used incineration techniques. However, a systematic study of the importance of upstream process techniques for the methane potential of pulp fibres is lacking. Therefore, biochemical methane potentials were determined at mesophilic conditions for 20 types of fibres processed by a variety of pulping and bleaching techniques and from different raw materials. This included fibres from kraft, sulphite, semi-chemical, chemical thermo-mechanical (CTMP) and thermo-mechanical pulping plants and milled raw wood. The pulping technique was clearly important for the methane potential, with the highest potential achieved for kraft and sulphite fibres (390–400 Nml CH4 g VS− 1 ). For raw wood and CTMP, hardwood fibres gave substantially more methane than the corresponding softwood fibres (240 compared to 50 Nml CH4 g VS− 1 and 300 compared to 160 Nml CH4 g VS− 1 , respectively). Nuclear magnetic resonance characterization of the organic content demonstrated that the relative lignin content of the fibres was an important factor for methane production, and that an observed positive effect of bleaching on the methane potential of softwood CTMP fibres was likely related to a higher degree of deacetylation and improved accessibility of the hemicellulose. In conclusion, fibres from kraft and sulphite pulping are promising substrates for methane production irrespective of raw material or bleaching, as well as fibres from CTMP pulping of hardwood.

Anaerobic digestion (AD) is an established process for integrating waste management with renewable energy and nutrient recovery. Much of the research in this field focuses on the utilisation of new substrates, yet their effects on operational aspects such as fluid behaviour and power requirement for mixing are commonly overlooked, despite their importance for process optimisation. This study analysed rheological characteristics of samples from 21 laboratory-scale continuous stirred-tank biogas reactors (CSTBRs) digesting a range of substrates, in order to evaluate substrate effect on mixing efficiency and power demand through computational fluid dynamics (CFD). The results show that substrate and process parameters, such as solids content and organic loading, all have a significant effect on CSTBR fluid rheology. The correlation levels between rheological and process parameters were different across substrates, while no specific fluid behaviour patterns could be associated with substrate choice. Substrate should thus be considered an equally important rheology effector as process parameters. Additional substrate-related parameters should be identified to explain the differences in correlations between rheological and process parameters across substrate groups. The CFD modelling revealed that the rheology differences among the AD processes have significant implications for mixing efficiency and power demand of the CSTBRs, highlighting the importance of considering the substrate-induced effects on CSTBR rheology before including a new substrate.

This study evaluates a simultaneous assessment of organic matter (OM) and trace elements (TE) bio-accessibility in substrate and digestate from a full-scale anaerobic digester by a sequential OM extraction method. Simultaneous release of TE was determined along with the extraction of different OM fractions and the effects of extracting reagents on characteristics of OM were evaluated by nuclear magnetic resonance (NMR) spectroscopy. The reagents used for sequential extraction of OM were not enough selective. However, proteins were particularly removed by 0.1 M NaOH, while 72% H2SO4 mainly extracted hemicellulose and cellulose. The OM fractionation allowed for simultaneous extraction of greater than60% of total As, Cd, Co, Fe, Mn, Ni and Zn, while the extraction was limited for Al, Cr, Cu, Mo, and Pb. In substrate, greater than50% of total As, Co, Mn and Ni and less than40% of total Fe, Zn and Mo were identified in bio-accessible fractions. In digestate, all elements demonstrated poor bio-accessibility except for As.less thanbr /greater than (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Anaerobic digestion (AD) is a biological process where a variety of microorganisms are the key factors for transforming complex organic structure into biogas, a mixture of methane, carbon dioxide, and other trace gases. Linked to the production of biogas is also the unique possibility to utilize and recycle nutrients released during the digestion. This gives the AD-process a key role in the development of many biorefinery concepts supporting industrial symbiosis and circular economy. The overall process efficiency is linked to the microbial steps which are affected by regulating factors including the characteristic of the substrate and the engineered process layout, together with the targeted outputs (raw biogas, compressed/liquid biogas, fertilizer, and/or other refined products). Between the digestion steps, the hydrolysis, the first microbial step, is often the rate-limiting step in degradation of polymeric substrates. As such, securing efficient and cost-effective hydrolysis represents today the key to process complex biomasses not yet fully utilized within the AD despite its high methane potential.

As a result of rapid development in China and the growth of megacities, large amounts of organic wastes are generated within relatively small areas. Part of these wastes can be used to produce biogas, not only to reduce waste-related problems, but also to provide renewable energy, recycle nutrients, and lower greenhouse gases and air polluting emissions. This article is focused on the conditions for biogas solutions in Guangzhou. It is based on a transdisciplinary project that integrates several approaches, for example, literature studies and lab analysis of food waste to estimate the food waste potential, interviews to learn about the socio-technical context and conditions, and life-cycle assessment to investigate the performance of different waste management scenarios involving biogas production. Xiaoguwei Island, with a population of about 250,000 people, was chosen as the area of study. The results show that there are significant food waste potentials on the island, and that all studied scenarios could contribute to a net reduction of greenhouse gas emissions. Several socio-technical barriers were identified, but it is expected that the forthcoming regulatory changes help to overcome some of them.

This study assesses the molecular characteristics of particulate organic matter (POM) in agricultural and food waste digesters and elucidates the molecular properties of the recalcitrant POM fraction, which remains in the digestate after AD process. Molecular properties of POM in influent (substrate) and effluent (digestate) of seven full-scale AD plants (three agricultural waste and four food waste digesters) were characterized and compared using solid-state ¹³C cross-polarization magic angle spinning (CP-MAS) and solution-state ¹H,¹³C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy. Comparison of the POM structural compositions of substrate and digestate from each AD plant revealed an enrichment of protein structures relative to the carbohydrates in most cases, implying a preferential degradation of the carbohydrates over proteins and/or increase of microbial biomass upon AD of agricultural and food wastes. Distinctive molecular structures of labile and recalcitrant fractions of POM, subjected to AD, were identified by comparing the NMR spectra of all substrate and digestate POM. Accordingly, the labile POM fraction in food and agricultural solid wastes is characterized by structural entities of lipids and starch-like carbohydrates, whereas recalcitrant POM structures resemble alkyl and aromatic subunits of amino acids, lignin, and polysaccharides with β-glycosidic linkages. This information serves as a basis to further explore optimization approaches for improving AD of the underutilized POM and the fate of organic matter in digestate-amended arable lands.

Background

Waste lipids are attractive substrates for co-digestion with primary and activated sewage sludge (PASS) to improve biogas production at wastewater treatment plants. However, slow conversion rates of long-chain fatty acids (LCFA), produced during anaerobic digestion (AD), limit the applicability of waste lipids as co-substrates for PASS. Previous observations indicate that the sulfide level in PASS digesters affects the capacity of microbial communities to convert LCFA to biogas. This study assessed the microbial community response to LCFA loads in relation to sulfide level during AD of PASS by investigating process performance and microbial community dynamics upon addition of oleate (C18:1) and stearate (C18:0) to PASS digesters at ambient and elevated sulfide levels.

Results

Conversion of LCFA to biogas was limited (30% of theoretical biogas potential) during continuous co-digestion with PASS, which resulted in further LCFA accumulation. However, the accumulated LCFA were converted to biogas (up to 66% of theoretical biogas potential) during subsequent batch-mode digestion, performed without additional substrate load. Elevated sulfide level stimulated oleate (but not stearate) conversion to acetate, but oleate and sulfide imposed a synergistic limiting effect on acetoclastic methanogenesis and biogas formation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea showed that differences in sulfide level and LCFA type resulted in microbial community alterations with distinctly different patterns. Taxonomic profiling of the sequencing data revealed that the phylum Cloacimonetes is likely a key group during LCFA degradation in PASS digesters, where different members take part in degradation of saturated and unsaturated LCFA; genus W5 (family Cloacimonadaceae) and family W27 (order Cloacimonadales), respectively. In addition, LCFA-degrading Syntrophomonas, which is commonly present in lipid-fed digesters, increased in relative abundance after addition of oleate at elevated sulfide level, but not without sulfide or after stearate addition. Stearate conversion to biogas was instead associated with increasing abundance of hydrogen-producing Smithella and hydrogenotrophic Methanobacterium.

Conclusions

Long-chain fatty acid chain saturation and sulfide level are selective drivers for establishment of LCFA-degrading microbial communities in municipal sludge digesters.

Knowledge of microbial community dynamics in relation to process perturbations is fundamental to understand and deal with the instability of anaerobic digestion (AD) processes. This study aims to investigate the microbial community structure and function of a thermophilic AD process, fed with a chemically defined substrate, and its association with process performance stability. Next generation amplicon sequencing of 16S ribosomal RNA (rRNA) genes revealed that variations in relative abundances of the predominant bacterial species, Defluviitoga tunisiensis and Anaerobaculum hydrogeniformans, were not linked to the process performance stability, while dynamics of bacterial genera of low abundance, Coprothermobacter and Defluviitoga (other than D. tunisiensis), were associated with microbial community function and process stability. A decrease in the diversity of the archaeal community was observed in conjunction with process recovery and stable performance, implying that the high abundance of specific archaeal group(s) contributed to the stable AD. Dominance of hydrogenotrophic Methanoculleus particularly corresponded to an enhanced microbial acetate and propionate turnover capacity, whereas the prevalence of hydrogenotrophic Methanothermobacter and acetoclastic Methanosaeta was associated with instable AD. Acetate oxidation via syntrophic interactions between Coprothermobacter and Methanoculleus was potentially the main methane-formation pathway during the stable process. We observed that supplementation of Se and W to the medium improved the propionate turnover by the thermophilic consortium. The outcomes of our study provided insights into the community dynamics and trace element requirements in relation to the process performance stability of thermophilic AD.

Sediments underlying hypoxic or anoxic water bodies constitute a net source of phosphorus to the bottom water. This source has the potential to enhance eutrophication. Benthic fluxes of dissolved phosphorus, iron and manganese were measured from hypoxic, normoxic, and normoxic bioturbated by the invasive polychaete Marenzelleria arctia sediment in a mesocosm experiment. The highest benthic phosphorus efflux was detected in mesocosms with the hypoxic treatment. Normoxic, bioturbated sediments led to weaker retention of phosphorus compared to oxic, defaunated sediments. Both iron and manganese fluxes increased under bioturbated conditions compared to defaunated sediments. This study shows that re-oxygenation of previously anoxic coastal sediments enhance phosphorus retention in the sediments. Colonisation by M. arctia induce strong mobilisation of iron and manganese due to its intense bioirrigation, which facilitates organic matter degradation and decreases the phosphorus retention by metal oxides in sediment.

This study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full-scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH3-N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse-transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH3-N, Fe, S, Mo and Ni. A co-occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy-sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.

Pretreatment of anaerobic digester samples by hydrochloric acid (HCl) resulted in removal of Fe-based mineral and coordination compounds, attenuating their interferences with solution-state nuclear magnetic resonance (NMR) spectroscopic characterization of the solid phase organic matter. Substrate (influent) and digestate (effluent) samples from two full-scale anaerobic digesters, designated CD (co-digester) and SSD (sewage sludge digester), were investigated. Pretreatment of CD samples with 0.2-2.0 mol l(-1) HCl and pretreatment of SSD samples with 1.0-3.0 mol l(-1) HCl removed 96-100% and 76-80% of total Fe, respectively. Pretreatment declined overall paramagnetic characteristics of digestate samples, manifested by 50% (CD) and 70% (SSD) decrease in electron paramagnetic resonance signal intensities. As a result, meaningful solution-state H-1,C-13 heteronuclear single quantum coherence and H-1 NMR spectra of DMSO-d(6) soluble organic matter could be acquired. Sample pretreatment with the lowest concentration of HCl resulted in alteration of C:N ratios in solid phase, likely due to removal of labile organic and inorganic C- and N-containing compounds, while elevating the HCl concentration did not further change the C:N ratios. Furthermore, sample pretreatment increased the solubility of carbohydrates and proteins in DMSO-d(6), enabling the detection of NMR resonances from certain structural units of carbohydrates (e.g. anomeric O2CH) and proteins (e.g. CH alpha in amino acids). Both attenuation of the paramagnetic matrix as well as art enhanced solubility of carbohydrate and protein fractions of the samples in DMSO-d(6) solvent contributed to an improved molecular characterization of anaerobic digester samples by solution-state NMR analysis.

Understanding fluid rheology is important for optimal design and operation of continuous stirred-tank biogas reactors (CSTBRs) and is the basis for power requirement estimates. Conflicting results have been reported regarding the applicability of total solid (TS) and/or total volatile solid (TVS) contents of CSTBR fluids as proxies for rheological properties. Thus, the present study investigates relationships between rheological properties of 12 full-scale CSTBR fluids, their substrate profiles, and major operational conditions, including pH, TS and TVS contents, organic loading rate, hydraulic retention time, and temperature. Rheology-driven power requirements based on various fluid characteristics were evaluated for a general biogas reactor setup. The results revealed a significant correlation only between the rheological fluid properties and TS or TVS contents for sewage sludge digesters and thermophilic co-digesters (CD), but not for mesophilic CD. Furthermore, the calculated power requirements for pumping and mixing, based on the various fluid characteristics of the studied CSTBRs, varied broadly irrespective of TS and TVS contents. Thus, this study shows that the TS and/or TVS contents of digester fluid are not reliable estimators of the rheological properties in CSTBRs digesting substrates other than sewage sludge.

This article deals with the interrelationship between overall chemical speciation of S, Fe, Co, and Ni in relation to metals bio-uptake processes in continuous stirred tank biogas reactors (CSTBR). To address this topic, laboratory CSTBRs digesting sulfur(S)-rich stillage, as well as full-scale CSTBRs treating sewage sludge and various combinations of organic wastes, termed co-digestion, were targeted. Sulfur speciation was evaluated using acid volatile sulfide extraction and X-ray absorption spectroscopy. Metal speciation was evaluated by chemical fractionation, kinetic and thermodynamic analyses. Relative Fe to S content is identified as a critical factor for chemical speciation and bio-uptake of metals. In reactors treating sewage sludge, quantity of Fe exceeds that of S, inducing Fe-dominated conditions, while sulfide dominates in laboratory and co-digestion reactors due to an excess of S over Fe. Under sulfide-dominated conditions, metals availability for microorganisms is restricted due to formation of metal-sulfide precipitates. However, aqueous concentrations of different Co and Ni species were shown to be sufficient to support metal acquisition by microorganisms under sulfidic conditions. Concentrations of free metal ions and labile metal complexes in aqueous phase, which directly participate in bio-uptake processes, are higher under Fe-dominated conditions. This in turn enhances metal adsorption on cell surfaces and bio-uptake rates.

Sweden has the ambition to increase its annual biogas production from the current level of 1.9 to 15 TWh by 2030. The unused capacity of existing anaerobic digesters at wastewater treatment plants is among the options to accomplish this goal. This study investigated the feasibility of utilizing the organic fraction of municipal solid waste (OFMSW) as a co-substrate, with primary and waste-activated sewage sludge (PWASS) for production of biogas, corresponding to 3:1 ratio on volatile solid (VS) basis. The results demonstrated that co-digestion of OFMSW with PWASS at an organic loading rate of 5 gVS l−1 day−1 has the potential to increase the biogas production approximately four times. The daily biogas production increased from 1.0 ± 0.1 to 3.8 ± 0.3 l biogasl−1 day−1, corresponding to a specific methane production of 420 ± 30 Nml methane gVS−1 during the laboratory experiment. Co-digestion of OFMSW with PWASS showed a 50:50 distribution of hydrogenotrophic and aceticlastic methanogens in the digester and enhanced the turnover kinetics of intermediate products (acetate, propionate, and oleate). Practical limitations potentially include the need for sludge dewatering to maintain a sufficient hydraulic retention time (17 days in this study), as well as additional energy consumption for mixing due to an increased sludge apparent viscosity (from 1.8 ± 0.1 to 45 ± 4.8 mPa*s in this study) at elevated OFMSW-loading rates.

The inhibitory effects of sulfide on microbial processes during anaerobic digestion have been widely addressed. However, other effects of sulfide are less explored, given that sulfide is a potential sulfur source for microorganisms and its high reactivity triggers a suit of abiotic reactions. We demonstrated that sulfide interaction with Fe regulates the dynamics and activities of microbial community during anaerobic digestion. This was manifested by the S:Fe molar ratio, whose increase adversely influenced the acetoclastic methanogens, Methanosaeta, and turnover of acetate. Dynamics of hydrogenotrophic methanogens, Methanoculleus and Methanobrevibacter, were presumably influenced by sulfide-induced changes in the partial pressure of hydrogen. Interestingly, conversion of the long-chain fatty acid (LCFA), oleate, to methane was enhanced together with the abundance of LCFA-degrading, beta-oxidizing Syntrophomonas at an elevated S:Fe molar ratio. The results suggested that sulfur chemical speciation is a controlling factor for microbial community functions in anaerobic digestion processes. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

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This study used semi-continuous laboratory scale biogas reactors to simulate the effects of trace-element addition in different combinations, while degrading the organic fraction of municipal solid waste and slaughterhouse waste. The results show that the combined addition of Fe, Co and Ni was superior to the addition of only Fe, Fe and Co or Fe and Ni. However, the addition of only Fe resulted in a more stable process than the combined addition of Fe and Co, perhaps indicating a too efficient acidogenesis and/or homoacetogenesis in relation to a Ni-deprived methanogenic population. The results were observed in terms of higher biogas production (+9%), biogas production rates (+35%) and reduced VFA concentration for combined addition compared to only Fe and Ni. The higher stability was supported by observations of differences in viscosity, intraday WA-and biogas kinetics as well as by the 16S rRNA gene and 16S rRNA of the methanogens.(c) 2015 Elsevier Ltd. All rights reserved.

Geochemical parameters and major ion concentrations from sediments of a freshwater lake in the town of angstrom tvidaberg, southeastern, Sweden, were used to identify the geochemical processes that control the water chemistry. The lake sediments are anoxic, characterized by reduced sulfur and sulfidic minerals. The hypothesis tested is that in sulfidic-anaerobic contaminated sediments, the presence of redox potential changes creates a favorable condition for sulfide oxidation, resulting in the release of potentially toxic metals. The acid volatile sulfide (AVS) contents ranged from 5.5mol/g to 16mol/g of dry sediment. Comparison of total mine tailing metals (Sigma mine tailing metals) with simultaneously extracted metals (SEM) in sediments indicates that up to 20% of the Sigma mine tailing metals are bound to the solid phase as AVS. Consequently, the AVS and SEM analysis classified all sediment samples as potentially toxic in terms of heavy metal concentrations (i.e., SEM to AVS ratio distribution>1). Evaluation of hydrogeochemical data suggests that calcite dissolution, iron (III) oxyhydroxysulfate mineral jarosite (H-jarosite) precipitation, hematite precipitation, and siderite precipitation are the most prevailing geochemical processes that control the geochemical interactions between the water column and sediment in a mine-impacted lake. The geochemical processes were verified and quantified using a chemical equilibrium modeling program, Visual MINTEQ, Ver 3.1, beta. The identified geochemical processes create an environment in which the characteristics of sulfate-rich waters and acidic-iron produce the geochemical conditions for acid mine drainage and mobilization of toxic metals. (c) 2015 SETAC

Optimal supply of trace elements (TE) is a prerequisite for microbial growth and activity in anaerobic digestion (AD) bioprocesses. However, the required concentrations and ratios of essential TE for AD biotechnologies strongly depend on prevailing operating conditions as well as feedstock composition. Furthermore, TE in AD bioreactors undergo complex physicochemical reactions and may be present as free ions, complex bound or as precipitates depending on pH, or on the presence of sulfur compounds or organic macromolecules. To overcome TE deficiency, various commercial mineral products are typically applied to AD processes. The addition of heavy metals poses the risk of overdosing operating systems, which may be toxic to microbial consortia and ultimately the environment. Adequate supplementation, therefore, requires appropriate knowledge not only about the composition, but also on the speciation and bioavailability of TE. However, very little is yet fully understood on this specific issue. Evaluations of TE typically only include the measurement of total TE concentrations but do not consider the chemical forms in which TE exist. Thus detailed information on bioavailability and potential toxicity cannot be provided. This review provides an overview of the state of the art in approaches to determine bioavailable TE in anaerobic bioprocesses, including sequential fractionation and speciation techniques. Critical aspects and considerations, including with respect to sampling and analytical procedures, as well as mathematical modeling, are examined. The approaches discussed in this review are based on our experiences and on previously published studies in the context of the COST Action 1302: European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies.

Codigesting fats, oils, and greases with municipal wastewater sludge can greatly improve biomethanerecovery at wastewater treatment facilities. Process loading rates of fats, oils, and greases have beenpreviously tested with little knowledge of the digester microbial community structure, and high transientfat loadings have led to long chain fatty acid (LCFA) accumulation and digester upsets. This studyutilized recently-developed quantitative PCR assays for syntrophic LCFA-degrading bacteria along with16S amplicon sequencing to relate changes in microbial community structure to LCFA accumulationduring transient loading increases to an anaerobic codigester receiving waste restaurant oil andmunicipal wastewater sludge. The 16S rRNA gene concentration of the syntrophic b-oxidizing genusSyntrophomonas increased to ~15% of the Bacteria community in the codigester, but stayed below 3% inthe control digester that was fed only wastewater sludge. Methanosaeta and Methanospirillum were thedominant methanogenic genera enriched in the codigester, and together comprised over 80% of theArchaea community by the end of the experimental period. Constrained ordination showed that changesin the codigester Bacteria and Archaea community structures were related to measures of digester performance.Notably, the effluent LCFA concentration in the codigester was positively correlated to thespecific loading rate of waste oil normalized to the Syntrophomonas 16S rRNA concentration. Specificloading rates of 0e1.5 1012 g VS oil/16S gene copies-day resulted in LCFA concentrations below 30 mg/g TS, whereas LCFA accumulated up to 104 mg/g TS at higher transient loading rates. Based on thecommunity-dependent loading limitations found, enhanced biomethane production from high loadingsof fats, oils and greases can be achieved by promoting a higher biomass of slow-growing syntrophicconsortia, such as with longer digester solids retention times. This work also demonstrates the potentialfor controlling the loading rate of fats, oils, and greases based on the analysis of the codigester communitystructure, such as with quantitative PCR measurements of syntrophic LCFA-degrading bacteriaabundance.

The effects of sulfide removal by addition of Fe on chemical speciation of Co and Ni and how it may affect the microbial metal uptake processes in biogas reactors were assessed. The influent Fe concentration was increased in a semi-continuous stirred tank biogas reactor fed with sulfur-rich stillage. Performance of the reactor, turnover kinetics of volatile carboxylic acids as well as changes in the chemical speciation of Co and Ni were investigated. The results demonstrated that approximately 95% decrease in gaseous hydrogen sulfide content of the biogas, which was caused by addition of Fe, had no apparent effects on methane production and process stability, while it enhanced the short-term turnover time of propionate. Sulfide removal decreased the overall solubility of Co and Ni partially by 1) lowering the formation of the dominant Co- and Ni-sulfide complexes in the aqueous phase and 2) by promoting processes such as adsorption and coprecipitation of Co and Ni with FeS(s). Combination of chemical speciation and bio-uptake models suggested that a higher concentration of free Co and Ni ions is achieved at low sulfide concentrations which favors the active bio-uptake of these metals. However, it was argued that the decrease in soluble metal concentrations, which was induced as a result of the addition of Fe, demotes potential diffusion-driven, passive metal uptake by microorganisms.

The objective of the present study was to assess major chemical reactions and chemical forms contributing to solubility and speciation of Fe(II), Co(II), and Ni(II) during anaerobic digestion of sulfur (S)-rich stillage in semi-continuous stirred tank biogas reactors (SCSTR). These metals are essential supplements for efficient and stable performance of stillage-fed SCSTR. In particular, the influence of reduced inorganic and organic S species on kinetics and thermodynamics of the metals and their partitioning between aqueous and solid phases were investigated. Solid phase S speciation was determined by use of S Kedge X-ray absorption near-edge spectroscopy. Results demonstrated that the solubility and speciation of supplemented Fe were controlled by precipitation of FeS(s) and formation of the aqueous complexes of Fe-sulfide and Fe-thiol. The relatively high solubility of Co (similar to 20% of total Co content) was attributed to the formation of compounds other than Co-sulfide and Co-thiol, presumably of microbial origin. Nickel had lower solubility than Co and its speciation was regulated by interactions with FeS(s) (e.g. co-precipitation, adsorption, and ion substitution) in addition to precipitation/dissolution of discrete NiS(s) phase and formation of aqueous Ni-sulfide complexes.

There are several ways to improve biogas production in anaerobic digestion processes and a number of strategies may be chosen. Increased organic loading in existing plants will in most cases demand the introduction of new substrate types. However, to substantially increase the Swedish biogas production new, large-scale biogas plants digesting new substrate types need to be established.

Better utilization of existing digester volumes can be linked to: 

• Increase of organic loading rates and/or reduced hydraulic retention time
• Optimizing the anaerobic microbial degradation by identifying rate-limitations, its causes and possible remedies such as:
• Nutrient and trace element balances
• Needs and availability of trace element
• Process design aiming at an increase of the active biomass (e.g. recirculation of reactor material, two stage processes)
• Process inhibition (enzymatically regulated product inhibition and toxicity)
• Improved pre-treatment to increase degradation rates and VS-reduction
• Mixing and rheology
• Better monitoring and control
• Co-digestion with more high-potential substrates

The present report reviews a number of fields that are linked to improvements in the biogas production process as based on the bullets above.

A well-working, active biomass is a prerequisite for efficient biogas production processes, why factors affecting microbial growth are crucial to obtain stable processes at the highest possible organic load/lowest possible hydraulic retention time.

The microorganisms need nutrients, i.e. carbon, nitrogen, phosphorus, calcium, potassium, magnesium and iron as well as trace elements such as cobalt, nickel, manganese, molybdenum, selenium and tungsten for growth. The need of nutrients and trace elements varies with the substrate digested, the organic loading rate, the process design (e.g. the reactor configuration, the degree of recirculation etc). In addition, the complexity of the chemical reactions controlling the bioavailability of the trace metals is wide, why optimal addition strategies for trace elements needs to be developed.

Substrates as food wastes, sewage sludge, cattle manure, certain energy crops and algae are good bases to obtain processes with good nutrient- and trace element balances. These kinds of substrates can often be implemented for “mono-substrate” digestion, while substrates dominated by carbohydrates or fats needs to be co-digested or digested in processes modified by  e.g. nutrient- and trace element additions, sludge recirculation, etc. Protein-rich substrates often include enough nutrients, but can give other process problems (see below).

Iron, cobalt and nickel are the nutrients/trace elements given most attention so far. However, molybdenum, selenium and tungsten have also, among others, been shown effective in different AD applications. The effects have, however, mainly been shown on turnover of VFAs and hydrogen (resulting in increased methane formation), while just a few studies have addressed their direct effect on rates of hydrolysis, protein-, fat- and carbohydrate degradation. Selenium- and cobalt-containing enzymes are known to be involved in amino acid degradation, while selenium and tungsten are needed in fat- and long chain fatty acid degradation. Enzymes active in hydrolysis of cellulose have been shown to be positively affected by cobalt, cupper, manganese, magnesium and calcium. This implies that trace element levels and availability will directly affect the hydrolysis rates as well as rates and degradation pathways for digestion of amino acids, long chain fatty acids and carbohydrates. However, their effect on hydrolysis seems neglected, why studies are needed to map the metals present in active sites and co-factors of enzymes mediating these primary reactions in AD. Further investigations are then needed to elucidate the importance of the identified metals on the different degradation steps of AD aiming at increased degradation rates of polymeric and complex substrates. It should also be noted that the degradation routes for amino acid degradation in AD-processes, factors governing their metabolic pathways, and how ATP is gained in the different pathways seem unknown. The different routes may result in different degradation efficiencies, why a deeper knowledge within this field is called for.

Trace metals added to biogas reactors have positive effects on the process only if they are present in chemical species suitable for microbial uptake. Interaction of biogenic sulfide with trace metals has been identified as the main regulator of trace metal speciation during AD. Fe, Co and Ni instantaneously form strong sulfide precipitates in biogas reactors but at the same time show very different chemical speciation features. The soluble fraction of Co widely exceeded the levels theoretically possible in equilibrium with inorganic sulfide. The high level of soluble Co is likely due to association with dissolved organic compounds of microbial origin. Fe and Ni speciation demonstrated a different pattern dominated by low solubility products of inorganic metal sulfide minerals, where their solubility was controlled mainly by the interactions with different dissolved sulfide and organic ligands. To our knowledge, the information about chemical speciation of other trace metals (Se, Mo, and W among others) and its effects on the bioavailability in anaerobic digestion environments is rare. Providing information on the metal requirements by processes linked to their bioavailability in biogas reactors is identified as a key knowledge needed for maximizing the effect of metals added to biogas reactors. Further research is also needed for development and design of proper metal additive solutions for application in full scale biogas plants. A practical approach is to supplement trace metals in specific chemical forms, which are either suitable for direct bio-uptake or will hamper undesirable and bio-uptake-limiting reactions (e.g. mineral precipitation).

Recirculation of reactor material as a way to enrich and maintain an active microbial biomass (and, thus, an increase in the substrate turnover rate) in tank reactors has been tested for digestion of fat within BRCs project DP6. The methane yield increased from 70 to 90% of the theoretical potential at a fat-loading rate of 1.5 g VS/L and day. The same strategy has been successful during digestion of fiber sludge from the pulp and paper industry, i.e. the recirculation has been crucial in establishment of low hydraulic retention times. Also degradation of sewage sludge (SS) would likely be improved by recirculation as the retention time of the solid SS is prolonged in such a system. However, this remains to be tested. The recirculation concept also needs to be evaluated in larger scale reactors to form a base to include extra costs and energy consumption vs. the benefits from increased yields.

To divide the anaerobic digestion process into two phases, where the hydrolytic/acidogenic and the syntrophic/methanogenic stages of anaerobic digestion are separated, might be a way to enhance degradation of lignocellulosic materials as the hydrolysis of these compounds may be inhibited by the release of soluble sugars. It should be noted that the natural AD of ruminates is phase-separated and improvements in AD can likely be achieved using these natural systems as a starting point. Also the degradation of aromatic and chlorinated species is likely enhanced by phase separation. One way to obtain such systems is to combine a leached bed for hydrolysis of insoluble material with a methanogenic reactor treating the leachate. Plug flow reactors might be another possibility as well as membrane reactors, which physically separates the hydrolyzing and methanogenic phases.

Inhibition caused by toxic levels of ammonia (protein- and ammonia rich substrates), fat-rich substrates and long chain fatty acids (LCFAs), aromatic compounds, salts etc. have been reported in many cases and some remedies are suggested. Ammonia can be stripped off as a measure to overcome too high levels. Another option is to adjust pH of the reactor liquid by addition of acid shifting the ammonia-ammonium balance in the system towards less free ammonia. A decrease in alkalinity by acid addition might also affect the availability of trace elements as solubility of trace metal mineral phases is generally higher at lower pH. LCFA degradation has been shown to benefit from periodic additions of fat and is, thus, an effective strategy to minimize inhibition by the release of the LCFA. Adsorption to zeolites has also been shown to abate the inhibition by LCFA. The best way to avoid inhibition is, however, to keep the processes nutritionally well balanced and using concepts suitable for the actual substrate mix digested (i.e. sludge recirculation, phase separation etc.) in order to obtain the highest possible degradation rate for problematic compounds, thus, avoiding accumulation of inhibitory components such as LCFA and aromatics. High ammonia and salt levels can often be regulated by the substrate mix.

The hydrolysis is often reported as rate limiting in digestion of complex polymers in balanced anaerobic digestion systems, while the methanogensis is regarded as rate-limiting for more easily degraded substrates. As mentioned above the effect on methane formation rates by the addition of trace elements have been shown in numerous studies, while their effect on the hydrolysis and acidogenic AD steps are much less studied. Thus, the effects of the trace elements on the early steps in the AD-chain need to be investigated further.

To obtain high-rate hydrolysis, effective and energy efficient pre-treatment methods are crucial for a large number of substrates. The rate of hydrolysis is to a large extent dependent on the properties of the organic compounds in the substrate e.g. carbohydrates, proteins, fat or lignocellulosic material as well as particle size and pre-treatment methods applied. The establishment and colonization by sessile microorganisms and biofilms is highly important for efficient and high rate hydrolysis. Microbial formation of organic compounds and the availability of surfaces are factors influencing these key processes, which in turn are tightly coupled to the growth conditions for the hydrolyzing microorganisms. This is an area recently brought up as an issue for detailed research.

Mixing is mostly needed for effective high-rate biogas production, but too extensive mixing can destroy the syntrohpic interactions necessarily taking place during AD. However, the efficiency of the mixing system design in relation to colonization, presences of dead zones, changes in viscosity/rheology, etc. seem unclear and this area thus calls for further attention. 

In high-loaded efficient processes a monitoring program following parameters e.g. organic loading rate, gas-production, VS-reduction, pH and VFA-levels is needed. This can be achieved through sampling and analysis off line, but there are of course benefits with on-line monitoring. A number of different methods have been suggested and tested, and some titration- and spectroscopic methods are applied, but none seems commonly in use. The reasons for the low interest to apply these methods may be the need for expertise on calibration, validation and multivariate analysis of most on-line methods, high maintenance demands (cost and time), and l functional problems related to fouling, gas bubbles, sensor location, disturbing particles etc.

New substrates with the highest potential for use in existing or new biogas plants seem to be forestry-based biomass, certain energy crops and macro-algae. Both the energy crops and the macro-algae can be chosen to give nutritionally well balanced AD-processes, while AD on forestry biomass demands nutrient supplements. For both the energy crops and the macro-algae sustainable cultivation systems need to be developed. Crop rotation systems should be employed to minimize tillage as well as fertilization- and pesticide utilization at highest possible TS-yields. System analyses aiming at sustainability and economy of TS and methane yields per ha including needs of nutrient supplements should therefore be performed.

In all three cases (forestry biomass, energy crops and algae) pre-treatment methods to create high internal surface areas are needed. However, the pre-treatment methods chosen need to be highly energy- and resource efficient to obtain sustainable systems (a positive energy balance). New plants will for profitability likely need to be large with highly developed infrastructure for substrates supply and distribution of the produced biogas/electricity nearby. Process concepts aiming at highest possible loading rates at shortest possible retention time will be needed, which likely are met by including both phase-separated process systems and systems for sludge recirculation.

It should also be noted that the lignin in substrates from forestry biomass needs to be used for production of e.g. polymeric materials or as a fuel to obtain reasonable energy balances for AD of lignocellulose. Pre-treatment methods obtaining separation of lignin is therefore needed. A substantial research and development is in progress within this field.

The possibilities for AD within the pulp and paper industry are interesting, especially if specific effluents within the pulp- and paper production units are selected and the raw material for the pulp and paper production is chosen considering the biogas yields of the residues.

We investigated the equilibrium chemistry and chemical speciation of S, Fe and metals (Co, Ni, Cu, Zn, Cd, and Pb) in eight full scale Continuous Stirred Tank Biogas Reactors (CSTBR). Five reactors were digesting mixtures of different organic wastes (referred to as Co-Digester; CD) and three were digesting Sewage Sludge (SS). Iron was continuously added to the CD reactors to remove sulfide produced during anaerobic digestion and SS was rich in Fe, amended for phosphate removal in wastewater treatment plants prior to anaerobic digestion. As a consequence of different S:Fe molar ratios (0.3–2.8), ferruginous (Fe(II)-dominated) conditions prevailed in SS reactors and sulfidic (S(-II)-dominated) conditions in CD reactors. In all reactors, the chemical speciation of S, as determined by S K-edge X-ray Absorption Near-Edge Structure spectroscopy, was dominated by FeS(s). Reduced organic S forms, consisting of RSH (thiol) and RSR (organic sulfide), were the second most abundant S species. Zero-valent S (elemental S, polysulfides, and possible traces of pyrite) was detected in all reactors, ranging between 6% and 26% of total S, with the highest proportion formed under ferruginous conditions. Thermodynamic modeling suggested that Fe in the aqueous phase was dominated by Fe(II)-thiol complexes under sulfidic conditions (CD reactors) and by Fe(II)-phosphate complexes under ferruginous conditions (SS reactors). Thiols, representing organic functional groups, and sulfide complexes were the major aqueous species of Co(II), Ni(II), Cd(II) and Pb(II) under sulfidic conditions. Under ferruginous conditions thiol complexes were still important, but carbonate and phosphate complexes in particular dominated the aqueous phase speciation of Co(II) and Ni(II). The aqueous phase speciation of Zn and Cu was dominated by Zn(II)-sulfide and Cu(I)-polysulfide complexes, respectively. The results highlights the importance of S:Fe molar ratio as a regulating factor for the chemical speciation of metals in biogas reactors which in turn is important for microbial trace metal uptake and growth as well as potential metal toxicity. Both these aspects are critical for a successful performance of biogas production process.

The microbial community of 21 full-scale biogas reactors was examined using 454 pyrosequencing of 16S rRNA gene sequences. These reactors included seven (six mesophilic and one thermophilic) digesting sewage sludge (SS) and 14 (ten mesophilic and four thermophilic) codigesting (CD) various combinations of wastes from slaughterhouses, restaurants, households, etc. The pyrosequencing generated more than 160 000 sequences representing 11 phyla, 23 classes, and 95 genera of Bacteria and Archaea. The bacterial community was always both more abundant and more diverse than the archaeal community. At the phylum level, the foremost populations in the SS reactors included Actinobacteria, Proteobacteria, Chloroflexi, Spirochetes, and Euryarchaeota, while Firmicutes was the most prevalent in the CD reactors. The main bacterial class in all reactors was Clostridia. Acetoclastic methanogens were detected in the SS, but not in the CD reactors. Their absence suggests that methane formation from acetate takes place mainly via syntrophic acetate oxidation in the CD reactors. A principal component analysis of the communities at genus level revealed three clusters: SS reactors, mesophilic CD reactors (including one thermophilic CD and one SS), and thermophilic CD reactors. Thus, the microbial composition was mainly governed by the substrate differences and the process temperature.

Addition of Co and Ni often improves the production of biogas during digestion of organic matter, i.e. increasing CH4-production, process stability and substrate utilization which often opens for higher organic loading rates (OLRs). The effect of Co and Ni addition was evaluated by measuring methane production, volatile solids reduction, pH and concentration of volatile fatty acids (VFAs). A series of six lab-scale semi-continuously fed biogas tank reactors were used for this purpose. The chemical forms and potential bioavailability of Co and Ni were examined by sequential extraction, acid volatile sulfide extraction (AVS) and simultaneously extracted metals. Furthermore, the sulfur speciation in solid phase was examined by sulfur X-ray absorption near edge structure spectroscopy. The effect of Co and Ni deficiency on the microbial community composition was analyzed using quantitative polymerase chain reaction and 454-pyrosequencing. The results showed that amendment with Co and Ni was necessary to maintain biogas process stability and resulted in increased CH4-production and substrate utilization efficiency. 10-20% of the total Co concentration was in dissolved form and should be regarded as easily accessible by the microorganisms. In contrast, Ni was entirely associated with organic matter/sulfides (mainly AVS) and regarded as very difficult to take up. Still Ni had stimulatory effects suggesting mechanisms such as dissolution of NiS to be involved in the regulation of Ni availability for the microorganisms. The microbial community structure varied in relation to the occurrence of Ni and Co. The acetate-utilizing Methanosarcinales dominated during stable process performance, i.e. when both Co and Ni were supplied, while hydrogenotrophic Methanomicrobiales increased together with VFA concentrations under Co or Ni deficiency. The increase was more pronounced at Co limitation. This study demonstrates that there are good possibilities to improve the performance of biogas processes digesting sulfur-rich substrates by supplementation of Co and Ni.

Nutrient reduction and the improvement of bottom water oxygen concentrations are thought to be key factors in the recovery of eutrophic aquatic ecosystems. The effects of reoxygenation and bioturbation of natural hypoxic sediments in the Baltic Sea were studied using a mesocosm experiment. Anoxic sediment box cores were collected from 100 m depth in Kanholmsfjärden (Stockholm Archipelago) and maintained in flow-through mesocosms with 3 treatments: (1) hypoxic: supplied with hypoxic water; (2) normoxic: supplied with oxic water; and (3) Marenzelleria: supplied with oxic water and the polychaete Marenzelleria spp. (2000 ind. m^–2). After a 7 wk long conditioning period, net fluxes of dissolved O₂, CH₄, Fe²⁺, Mn²⁺, NH₄⁺, NO₂^-, NO₃^-, PO₄^3- and H₄SiO₄, and rates of nitrate ammonification (DNRA), denitrification and anammox were determined. Phosphate was taken up by the sediment in all treatments, and the uptake was highest in the normoxic treatment with Marenzelleria. Normoxic conditions stimulated the denitrification rate by a factor of 5. Denitrification efficiency was highest under normoxia (50%), intermediate in bioturbated sediments (16%), and very low in hypoxic sediments (4%). The shift from hypoxic to normoxic conditions resulted in a significantly higher retention of NH₄⁺, H₄SiO₄ and Mn²⁺ in the sediment, but the bioturbation by Marenzelleria reversed this effect. Results from our study suggest that bioturbation by Marenzelleria stimulates the exchange of solutes between sediment and bottom water through irrigation and enhances bacterial sulfate reduction in the burrow walls. The latter may have a toxic effect on nitrifying bacteria, which, in turn, suppresses denitrification rates.

Several previous studies reported stimulatory effects on biogas process performance after trace metal supplementation. However, the regulation of the bioavailability in relation to chemical speciation, e.g. the role of sulfide is not fully understood. The objective of the present study was to determine the effect of sulfide on chemical speciation and bioavailability of Co and Ni in lab-scale semicontinuous stirred biogas tank reactors treating stillage. The chemical forms and potential bioavailability of Co and Ni were studied by sequential extraction, analysis of acid-volatile sulfide (AVS), and simultaneously extracted metals. The results demonstrated that Ni was completely associated to the organic matter/sulfide fraction and AVS, suggesting low potential bioavailability. Cobalt was predominantly associated to organic matter/sulfide and AVS, but also to more soluble fractions, which are considered to be more bioavailable. Process data showed that both Co and Ni were available for microbial uptake. Although the actual bioavailability of Co could be explained by association to more bioavailable chemical fractions, the complete association of Ni with organic matter/sulfides and AVS implies that Ni was taken up despite its expected low bioavailability. It was concluded that extensive Co- and Ni-sulfide precipitation did not inhibit microbial uptake of Co and Ni in the reactors.

Dissolved organic matter (DOM) was characterized in eight full scale continuous stirred tank biogas reactors (CSTBR) using solid-phase extraction and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). An overview of the DOM molecular complexity in the samples from biogas reactors with conventional operational conditions and various substrate profiles is provided by assignments of unambiguous exact molecular formulas for each measured mass peak. Analysis of triplicate samples for each reactor demonstrated the reproducibility of the solid-phase extraction procedure and ESI-FT-ICR-MS which allowed precise evaluation of the DOM molecular differences among the different reactors. Cluster analysis on mass spectrometric data set showed that the biogas reactors treating sewage sludge had distinctly different DOM characteristics compared to the codigesters treating a combination of organic wastes. Furthermore, the samples from thermophilic and mesophilic codigesters had different DOM composition in terms of identified masses and corresponding intensities. Despite the differences, the results demonstrated that compositionally linked organic compounds comprising 28-59% of the total number of assigned formulas for the samples were shared in all the reactors. This suggested that the shared assigned formulas in studied CSTBRs might be related to common biochemical transformation in anaerobic digestion process and therefore, performance of the CSTBRs.

Rheological properties of reactor liquids are important for the construction and intensity mixing systems in biogas reactors. Most frequently the total solids content (TS) is used as a proxy to guide in these matters. In order to establish a more comprehensive basis the rheology of twelve full-scale continuously stirred tank biogas reactors was characterized and related to differences in substrate composition and operational conditions. Reactor material from eight mesophilic (36−38°C) and four thermophilic (52−55°C) reactors were sampled at two occasions. The feedstocks of nine of these reactors were included in the analysis. Two of the mesophilic and one of the thermophilic digesters were fed sewage sludge (SS), while the others digested mixtures of organic matter including slaughterhouse waste (SHW), food industry waste (FIW), fat, manure, fodder residues and the organic fraction of municipal solid waste (OFMSW). The organic loading rates ranged 2.4−3.6 kg VS m^-3 d^-1 and the TS of the reactor materials were at 1.8−5.3% . The rheological characteristics of the reactor liquids were interpreted from flow- and viscosity curves as well as from determination of dynamic viscosity, limit viscosity, yield stress, flow behavior and consistency index.

The fluid dynamic- and limit viscosities of the fluids ranged 5−600 mPa*s and 4−40 mPa*s, respectively. All reactor fluids except one from a thermohilic CD-reactor showed pseudoplastic behavior, since they became thinner with increasing shear stress until the viscosity reached a plateau of limit viscosity. In addition the mesophilic CD reactors were strongly thixothropic, i.e. they exhibited partial structure recovery. The results from the analysis of the thermophilic CD-reactors indicated a weak dilatant behavior, i.e. shear thickening behavior.

The results showed differences in viscosity despite similar TS-content for several reactor liquids. From this survey it is clear that the TS content of biogas reactor fluids is not a good estimator of the fluid viscosity and that the fluid characteristics vary as a result of substrate composition and process operation conditions.