Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Mentalization-oriented psychodynamic group therapy for patients with personality disorders: a naturalistic prospective cohort study.

OBJECTIVES: Patients with personality disorders (PDs) are often treated with non-manualized psychodynamic group therapy (PDT) lasting for several years. Non-manualized PDT often combines a variety of therapeutic approaches from different PDT traditions, including mentalization-based therapy. Currently, little is known about the effect of this long-term, costly treatment. This study investigated the extent to which patients with different PDs benefit from mentalization-oriented PDT as it is implemented in clinical practice in terms of symptom severity, interpersonal problems, and general functioning. METHODS: The design was a naturalistic, prospective cohort study. Seventy-five consecutive PD patients were assessed before treatment with the Symptom Checklist-90 Revised (SCL-90-R) as the primary outcome measure and the Inventory of Interpersonal Problems (IIP) and Global Assessment of Functioning (GAF) as secondary outcome measures. The sample was repeatedly assessed every 12 months for up to 36 months. Paired t-tests were applied to examine the effectiveness of the intervention. RESULTS: Among completers (n = 42; 56%), improvement was observed on the SCL-90-R: Global Severity Index (mean change = -0.45 [95% CI = -0.72, -0.19]; Cohen's d = -0.55), Positive Symptom Distress Index (-0.40 [-0.63, -0.17]; -0.56); Positive Symptoms Total (-10.70 [-17.31, -4.09]; -0.52). Secondary outcomes also improved: IIP-total (mean change = -0.50 [95%CI = -0.74, -0.25]; Cohen's d = -0.66); GAF-Functioning (8.79 [6.32, 11.27]; 1.15); and GAF-Symptoms (10.67 [8.09, 13.25]; 1.34). CONCLUSIONS: Completers improved on symptom severity, interpersonal problems, and general functioning, with within-group effect sizes ranging from medium to large. Approximately half the sample dropped out, suggesting that mentalization-oriented PDT spanning several years may be unrealistic for many patients with PD. Significant outcomesThere are no clear guidelines for psychological interventions targeting personality disorders (PDs), and currently eclectic and non-manualized psychodynamic approaches lasting for up to 3 years are prevailing in some clinical practices.Although this treatment approach may have an effect on compliant patients, the high drop-out rate indicates that it may not be suitable for a large proportion of PD patients since it requires long-term commitment. Furthermore, it is difficult to identify the content of the non-manualized psychodynamic therapy and what helps the patients.More specific clinical guidelines emphasizing the application of evidence-based treatments or at least manualized treatments are warranted for the treatment of emotionally unstable PDs and other PDs. LimitationsThe naturalistic study design, without any control group, limits conclusions about mechanisms of action of the intervention.Since the intervention was not manualized, it is unknown exactly which treatment was actually administered, which reduces external validity.The outcomes are based on completer data of a relatively small sample size with high drop-out rate.

Viscosity dynamics and the production of extracellular polymeric substances and soluble microbial products during anaerobic digestion of pulp and paper mill wastewater sludges.

The production processes of the pulp and paper industry often run in campaigns, leading to large variations in the composition of wastewaters and waste sludges. During anaerobic digestion (AD) of these wastes, the viscosity or the production of extracellular polymeric substances (EPS) and soluble microbial products (SMP) may be affected, with the risk of foam formation, inefficient digester mixing or poor sludge dewaterability. The aim of this study was to investigate how viscosity and production of EPS and SMP during long-term AD of pulp and paper mill sludge is affected by changes in organic loading rate (OLR) and hydraulic retention time (HRT). Two mesophilic lab-scale continuous stirred tank reactors (CSTRs) were operated for 800 days (R1 and R2), initially digesting only fibre sludge, then co-digesting fibre sludge and activated sludge. The HRT was lowered, followed by an increase in the OLR. Reactor fluids were sampled once a month for rheological characterization and analysis of EPS and SMP. The production of the protein fraction of SMP was positively correlated to the OLR, implicating reduced effluent qualities at high OLR. EPS formation correlated with the magnesium content, and during sulphate deficiency, the production of EPS and SMP increased. At high levels of EPS and SMP, there was an increase in viscosity of the anaerobic sludges, and dewatering efficiency was reduced. In addition, increased viscosity and/or the production of EPS and SMP were important factors in sludge bulking and foam formation in the CSTRs. Sludge bulking was avoided by more frequent stirring.

Substrate and operational conditions as regulators of fluid properties in full-scale continuous stirred-tank biogas reactors - implications for rheology-driven power requirements.

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.

Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design.

The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

Anaerobic digestion of alkaline bleaching wastewater from a kraft pulp and paper mill using UASB technique.

Anaerobic digestion of alkaline kraft elemental chlorine-free bleaching wastewater in two mesophilic, lab-scale upflow anaerobic sludge bed reactors resulted in significantly higher biogas production (250±50 vs. 120±30 NmL g [Formula: see text]) and reduction of filtered total organic carbon (fTOC) (60±5 vs. 43±6%) for wastewater from processing of hardwood (HW) compared with softwood (SW). In all cases, the gas production was likely underestimated due to poor gas separation in the reactors. Despite changes in wastewater characteristics, a stable anaerobic process was maintained with hydraulic retention times (HRTs) between 7 and 14 h. Lowering the HRT (from 13.5 to 8.5 h) did not significantly affect the process, and the stable performance at 8.5 h leaves room for further decreases in HRT. The results show that this type of wastewater is suitable for a full-scale implementation, but the difference in methane potential between SW and HW is important to consider both regarding process dimensioning and biogas yield optimization.

Cyanobacteria as a source of hydrogen for methane formation.

In a study during the 1970s co-variation of nitrogenase activity and methane formation associated with Sphagnum riparium was observed. This was suggested as evidence for a possible mechanism of hydrogen transfer from cyanobacteria to methanogens. We show experimentally that such a pathway is feasible. In a series of laboratory experiments, using a hydrogenase deficient strain of the heterocystous cyanobacterium Nostoc punctiforme and the hydrogenotrophic methanogen Methanospirillum hungateii in co-cultures, increasing light intensities resulted in elevated nitrogenase activity and methane production. The increase in methane production can be directly deduced from the nitrogenase activity of the N. punctiforme based on hydrogen balance calculations. These experimental results clearly suggest the possible existence of a novel photosynthetically regulated pathway for methane formation.

Sulfide in engineered methanogenic systems - Friend or foe?

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.

Ligand selectivity hotspots in serotonin GPCRs.

Serotonin is a neurotransmitter regulating numerous physiological processes also modulated by drugs, for example, schizophrenia, depression, migraine, and obesity. However, these drugs typically have adverse effects caused by promiscuous binding across 12 serotonin and more than 20 homologous receptors. Recently, structures of the entire serotonin receptor family uncovered molecular ligand recognition. Here, we present a map of 19 'selectivity hotspots', that is, nonconserved binding site residues governing selectivity via favorable target interactions or repulsive 'off-target' contacts. Furthermore, we review functional rationale from observed ligand-binding affinities and mutagenesis effects. Unifying knowledge underlying specific probes and drugs is critical toward the functional characterization of different receptors and alleviation of adverse effects.

Characterization of dissolved organic matter in full scale continuous stirred tank biogas reactors using ultrahigh resolution mass spectrometry: a qualitative overview.

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.

Anaerobic treatment of activated sludge from Swedish pulp and paper mills--biogas production potential and limitations.

The methane potential of activated sludge from six Swedish pulp and paper mills was evaluated. The methane production potential of sludge samples ranged from 100-200 NmL CH4 g(-1) volatile solids (VS) and for four of the six sludge samples the potential exceeded 170 NmL CH4 g(-1) VS. The effects of sludge age and dewatering on the methane production potential were evaluated. The effects of enzymatic and ultrasonic pre-treatment on the digestibility of sludge were also investigated, but energy or enzyme inputs in viable ranges did not exert a detectable, positive effect. Long-term, semi-continuous trials with sludge from two of the mills were also conducted in attempts to develop stable biogas production at loading rates up to 4 g VS L(-1). Cobalt addition (0.5 mg L(-1)) was here found to positively affect the turnover of acetate. High viscosity was a problem in all the experimental reactors and this limited the organic loading rate.

Transformation of methyltin chlorides and stannic chloride under simulated landfill conditions.

There is increasing concern regarding the fate of methyltins in the environment, particularly since large amounts of polyvinyl chloride (PVC) plastics are deposited in landfills. The potential transformation of methyltin chlorides and stannic chloride in landfills was investigated, by incubating the target substances at concentrations relevant to landfill conditions (100 and 500 µg Sn L(-1)). The amounts of methane formed in all treatment bottles, and controls, were measured to evaluate the general microbial activity of the inocula and possible effects of methyltins on the degradation of organic matter. The methyltins and stannic chloride were found to have no significant inhibitory effects on the activity of landfill micro-organisms, and the methanol used to disperse the tin compounds was completely degraded. In some experimental bottles, the methanol degradation gave rise to larger methane yields than expected, which was attributed to enhanced degradation of the waste material. Alkyltin analyses showed that monomethyltin trichloride at an initial concentration of 500 µg Sn L(-1) promoted methylation of inorganic tin present in the inoculum. No methylation activities were detected in the incubations with 100 µg Sn L(-1) methyltin chlorides (mono-, di- or tri-methyltin), but demethylation occurred instead. Levels of soluble inorganic tin increased during the incubation period, due partly to demethylation and partly to a release of tin from the waste inocula.

Virtual Screening and Design with Machine Intelligence Applied to Pim-1 Kinase Inhibitors.

Ligand-based virtual screening of large compound collections, combined with fast bioactivity determination, facilitate the discovery of bioactive molecules with desired properties. Here, chemical similarity based machine learning and label-free differential scanning fluorimetry were used to rapidly identify new ligands of the anticancer target Pim-1 kinase. The three-dimensional crystal structure complex of human Pim-1 with ligand bound revealed an ATP-competitive binding mode. Generative de novo design with a recurrent neural network additionally suggested innovative molecular scaffolds. Results corroborate the validity of the chemical similarity principle for rapid ligand prototyping, suggesting the complementarity of similarity-based and generative computational approaches.

Effect of Cobalt, Nickel, and Selenium/Tungsten Deficiency on Mesophilic Anaerobic Digestion of Chemically Defined Soluble Organic Compounds.

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 °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.

Molecular characterization of particulate organic matter in full scale anaerobic digesters: An NMR spectroscopy study.

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 ß-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.

Rational design of antimicrobial C3a analogues with enhanced effects against Staphylococci using an integrated structure and function-based approach.

The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.

The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites.

Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.

Dynamics of a Perturbed Microbial Community during Thermophilic Anaerobic Digestion of Chemically Defined Soluble Organic Compounds.

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.

Microbial rRNA gene expression and co-occurrence profiles associate with biokinetics and elemental composition in full-scale anaerobic digesters.

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.

Anaerobic digestion of alfalfa silage with recirculation of process liquid.

Process liquid recirculation initially stimulated one-phase anaerobic digestion of alfalfa silage in two semi-continuously fed and stirred tank reactors. Thus, with increased pH, alkalinity and stability it was possible to increase the organic loading rate to 3 g VS L(-1) d(-1), as compared to 2.25 g VS L(-1) d(-1) in a control reactor without recirculation. However, the recirculation of liquid eventually caused an accumulation of organic and inorganic substances, leading to an inhibition of hydrolysis and methanogenesis. This inhibition of microbial activity was prevented in one of the processes by replacing 50% of the recirculated process liquid with water during the second half of the operation period. A multiple linear regression model of principal components using seven input variables explained the variance in output variables nearly as well as the original model using all 23 measured input variables. The results show that it is necessary to adjust the degree of liquid recirculation to reach an optimal process.