Complete genome sequence of Citroniella saccharovorans DSM 29873, isolated from human fecal sample.

A complete genome was recovered from Citroniella saccharovorans, strain DSM 29873, using Oxford Nanopore Technologies. The genome assembly contains 1,413,868 bp with 30.23% G+C content. The species belongs to the family Peptoniphilaceae and, as of yet, is the only cultivated representative of the genus Citroniella.

Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions.

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L-1 day-1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol-1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems.

Miniphocaeibacter halophilus sp. nov., an ammonium-tolerant acetate-producing bacterium isolated from a biogas system.

An anaerobic bacterial strain, designated AMB_01T, recovered from mesophilic propionate enrichment of a high-ammonia biogas digester, was characterised using phenotypic and molecular taxonomic methods. Cells of AMB_01T are coccus-shaped and often occur arranged as diplococci or sarcina. Growth occurred at 20-45 °C, initial pH 5.5-8.5 and with up to 0.7 M NH4Cl, with optimum growth at 37-42 °C and pH 8.0. AMB_01T achieved high cell density and highest acetate production when grown on carbohydrates, including monomers, disaccharides and polysaccharides, such as glucose, maltose, cellobiose and starch. The strain was also able to use amino acids and some organic acids and alcoholic compounds for growth. Acetate was formed as the main product and yeast was not required for growth. The major cellular fatty acids were summed feature 4 (iso-C17 : 1I and/or anteiso-C17 : 1B), C18 : 1ω7, C14 : 0, C16 : 0 and summed feature 3 (C16 : 1ω7 and/or iso-C15 : 0 2OH). The highest 16S rRNA gene sequence similarity found was with Miniphocaeibacter massiliensis (96.6 %), within the family Peptoniphilaceae, phylum Bacillota (Firmicutes). The genomic DNA G+C content was 29.0 mol%. An almost complete set of genes for the acetyl-CoA pathway was found. Genome comparisons between AMB_01T and close relatives showed highest digital DNA-DNA hybridisation to Finegoldia magna (23 %), highest average nucleotide identity with genome nucleotide and amino acid sequences to M. massiliensis (72 and 73 %, respectively) and highest average nucleotide identity (87 %) with Schnuerera ultunensis, indicating that AMB_01T represents a novel species. Analysis of genomic, chemotaxonomic, biochemical and physiological data confirmed that strain AMB_01T represents a novel species, for which the name Miniphocaeibacter halophilus sp. nov. is proposed. The type strain is AMB_01T (=DSM 110247T=JCM 39107 T).

Syntrophic propionate-oxidizing bacteria in methanogenic systems.

The mutual nutritional cooperation underpinning syntrophic propionate degradation provides a scant amount of energy for the microorganisms involved, so propionate degradation often acts as a bottleneck in methanogenic systems. Understanding the ecology, physiology and metabolic capacities of syntrophic propionate-oxidizing bacteria (SPOB) is of interest in both engineered and natural ecosystems, as it offers prospects to guide further development of technologies for biogas production and biomass-derived chemicals, and is important in forecasting contributions by biogenic methane emissions to climate change. SPOB are distributed across different phyla. They can exhibit broad metabolic capabilities in addition to syntrophy (e.g. fermentative, sulfidogenic and acetogenic metabolism) and demonstrate variations in interplay with cooperating partners, indicating nuances in their syntrophic lifestyle. In this review, we discuss distinctions in gene repertoire and organization for the methylmalonyl-CoA pathway, hydrogenases and formate dehydrogenases, and emerging facets of (formate/hydrogen/direct) electron transfer mechanisms. We also use information from cultivations, thermodynamic calculations and omic analyses as the basis for identifying environmental conditions governing propionate oxidation in various ecosystems. Overall, this review improves basic and applied understanding of SPOB and highlights knowledge gaps, hopefully encouraging future research and engineering on propionate metabolism in biotechnological processes.

Enrichment and description of novel bacteria performing syntrophic propionate oxidation at high ammonia level.

Inefficient syntrophic propionate degradation causes severe operating disturbances and reduces biogas productivity in many high-ammonia anaerobic digesters, but propionate-degrading microorganisms in these systems remain unknown. Here, we identified candidate ammonia-tolerant syntrophic propionate-oxidising bacteria using propionate enrichment at high ammonia levels (0.7-0.8 g NH3 L-1 ) in continuously-fed reactors. We reconstructed 30 high-quality metagenome-assembled genomes (MAGs) from the propionate-fed reactors, which revealed two novel species from the families Peptococcaceae and Desulfobulbaceae as syntrophic propionate-oxidising candidates. Both MAGs possess genomic potential for the propionate oxidation and electron transfer required for syntrophic energy conservation and, similar to ammonia-tolerant acetate degrading syntrophs, both MAGs contain genes predicted to link to ammonia and pH tolerance. Based on relative abundance, a Peptococcaceae sp. appeared to be the main propionate degrader and has been given the provisional name "Candidatus Syntrophopropionicum ammoniitolerans". This bacterium was also found in high-ammonia biogas digesters, using quantitative PCR. Acetate was degraded by syntrophic acetate-oxidising bacteria and the hydrogenotrophic methanogenic community consisted of Methanoculleus bourgensis and a yet to be characterised Methanoculleus sp. This work provides knowledge of cooperating syntrophic species in high-ammonia systems and reveals that ammonia-tolerant syntrophic propionate-degrading populations share common features, but diverge genomically and taxonomically from known species.

Metabolic performance of anaerobic digestion of chicken manure under wet, high solid, and dry conditions.

The anaerobic digestion (AD) of chicken manure as a solo substrate has been challenging due to the ammonium inhibition effects when adopting a high organic loading rate (OLR). In this study, through increasing both the total solid in the feeding materials from 5% to 20%, and the OLR from 1.7 to 7.1 g-volatile solids (VS)/(L·d), the AD of chicken manure under wet, high solid, and dry conditions, with a fixed hydraulic retention time of 20 days, was investigated. The results obtained indicated that the wet AD system could achieve a methane yield of 0.28 L/g-VS and a low volatile fatty acid level. However, the process deteriorated under dry conditions, and methane formed mainly through acetate oxidation and methanogenesis. Methanosarcina and Methanoplasma were found to be more tolerant But, whether the dry AD of chicken manure can survive an ammonia-stressed environment when the OLR is lowered, still needs investigation.

Inoculum Source Determines Acetate and Lactate Production during Anaerobic Digestion of Sewage Sludge and Food Waste.

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

Enhanced methanogenic performance and metabolic pathway of high solid anaerobic digestion of chicken manure by Fe2+ and Ni2+ supplementation.

High solid anaerobic digestion (AD) is a promising technology for the treatment of organic waste in terms of efficient bioenergy production and digestate volume reduction. However, the high solid AD of chicken manure has been a challenge due to the inhibition effects caused by high ammonia levels. In this study, the addition of 280 mg/L Fe2+ and 2 mg/L Ni2+ has been proven to enhance the microbial activity and to overcome inhibition under the stressed environment. The mesophilic digesters feeding with total solid around 150 g/kg operated at an organic loading rate of 4.8 g/(L d) based on volatile solid and a 20-day hydraulic retention time. The total ammonia-nitrogen reached around 6.8 g/L in control and Fe-Ni digesters. The results from the 147-day continuously-fed experiment provided an increment of 34% methane production and a 29% reduction of volatile fatty acids against control. The acetoclastic and hydrogenotrophic methanogenic activity was increased by 89% and 40% respectively. The high throughout sequencing results showed an increased percentage of Methanosarcina sp., which may have contributed to the shifting of the methanogenic pathway towards acetoclastic methanogenesis. The positive effects of Fe2+ and Ni2+ supplementation obtained in this study lay the foundation for its use in AD of nitrogen rich materials.

Growth Characteristics and Thermodynamics of Syntrophic Acetate Oxidizers.

Syntrophic acetate oxidation (SAO) plays a pivotal role in biogas production processes when aceticlastic methanogens are inhibited. Despite the importance of SAO, the metabolic interactions and syntrophic growth of the organisms involved are still poorly understood. Therefore, we studied growth parameters and interactions within constructed defined cocultures comprising the methanogen Methanoculleus bourgensis and one, or several, of the syntrophic acetate oxidizers Syntrophaceticus schinkii, [ Clostridium] ultunense, and Tepidanaerobacter acetatoxydans and a novel, uncharacterized bacterium. Cultivation experiments in a design-of-experiment approach revealed positive effects on methane production rate of increased ammonium levels (up to 0.2 M), temperature (up to 45 °C), and acetate concentrations (0.15-0.30 M). Molecular analyses and thermodynamic calculations demonstrated close interlinkages between the microorganisms, with available energies of -10 kJ/mol for acetate oxidation and -20 kJ/mol for hydrogenotrophic methanogenesis. The estimated generation time varied between 3 and 20 days for all syntrophic microorganisms involved, and the acetate minimum threshold level was 0.40-0.45 mM. The rate of methanogenesis depended on the SAO bacteria present in the culture. These data are beneficial for interpretation of SAO prevalence and competiveness against aceticlastic methanogens in anaerobic environments.

Enhancing anaerobic digestion of dairy and swine wastewater by adding trace elements: evaluation in batch and continuous experiments.

Trace elements play a critical role for microbial activity in anaerobic digestion (AD) but their effects were probably overestimated in batch tests and should be comparably evaluated in continuous systems. In this study, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ were added in different concentrations to manure wastewater, and the effects were compared in both batch and continuous systems. The results were used to demonstrate suitable trace element compositions for AD of dairy and swine wastewater, and to compare the outcomes from batch and continuous systems. Fe2+ and Zn2+ were identified as being the most efficient stimulant of dairy and swine wastewater respectively. The addition of 5 mg/L Fe2+ and 0.4 mg/L Zn2+ increased the batch specific methane yield by 62% and 126% for dairy and swine wastewater, respectively. Nevertheless, a lower increment of 2% and 21%, for dairy and swine wastewater was obtained in the 120-day continuously-fed experiments. The 16S rRNA gene sequencing results indicated a relationship between the methanogens population, specific methanogenic activities, propionate, and dissolved hydrogen. Conclusively, the addition of a low dosage of Fe2+ and Zn2+ is a feasible strategy to enhance the methanogenic metabolism of the AD of dairy and swine wastewater respectively.

The correlation of methanogenic communities' dynamics and process performance of anaerobic digestion of thermal hydrolyzed sludge at short hydraulic retention times.

Requirement of a long hydraulic retention time (HRT) for efficient degradation restrains the anaerobic digestion of hydrothermal pretreated sludge. Shortening the HRT can increase the treatment capacity of a plant but may also induce digester instability. This study investigated the impact of HRT on process performance and microbial community by consecutively operating a reactor for 145 days. The HRT was gradually decreased from 20 to 10, 5, and 3 days. The methane yield declined from 0.28 to 0.12 L/g-VSin with this shortening, and acetate concentration increased from 38 to 376 mg/L. Methanoculleus (58%) dominated methanogens at a 20 days HRT. However, the methanogenic structure shifted toward an increased level of Methanospirillum, representing 95% of the total archaea at a 3 days HRT. Microorganisms were almost washed out at the end of experiment. Conclusively, shortening HRTs is a feasible strategy to increase treatment capacity and produce more biogas at existing plants.

An explanation of the methanogenic pathway for methane production in anaerobic digestion of nitrogen-rich materials under mesophilic and thermophilic conditions.

The impact of temperature on the anaerobic digestion of chicken manure was investigated by studying the process performance and pathway for continuously-fed digesters under mesophilic and thermophilic conditions. The mesophilic digester obtained a 15% higher methane yield compared with the thermophilic digester. Mesophilic and thermophilic digester had free ammonia of 31 and 145 mg/L, respectively. The stable carbon isotope analysis indicated that 41% and 50% of acetate was converted to methane through the syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway under mesophilic and thermophilic conditions, respectively. The genus Pseudomonas represented 10% and 16% under mesophilic and thermophilic conditions, respectively. A high abundance of the methanogens genus Methanoculleus (94% of total methanogens) in mesophilic and the genus Methanothermobacter (96%) in thermophilic digesters indicated they were the main hydrogenotrophic partners in SAO. The present study therefore illustrated that methanogenic pathway shifting, induced by free ammonia, closely correlated to the process performance.

Impact of temperature and substrate concentration on degradation rates of acetate, propionate and hydrogen and their links to microbial community structure.

The present study investigates the conversion of acetate, propionate and hydrogen consumption linked to the microbial community structure and related to temperature and substrate concentration. Biogas reactors were continuously fed with coffee powder (20 g-COD/L) or acetate (20, 40, and 60 g-COD/L) and operated for 193 days at 37 °C or 55 °C conditions. Starting HRT was 23 days which was then reduced to 7 days. The kinetics of acetate and propionate degradation and hydrogen consumption rates were measured in batch assays. At HRT 7 days, the degradation rate of propionate was higher in thermophilic batches, while acetate degradation rate was higher at mesophilic conditions. The gaseous hydrogen consumption in acetate reactors increased proportionally with temperature and substrate concentration, while the dissolved hydrogen was not affected. The relative high abundance of hydrogentrophic methanogens indicated that the methanogenesis was directed towards the syntrophic acetate oxidation pathway at high acetate concentration and high temperature.

Detection of novel syntrophic acetate-oxidizing bacteria from biogas processes by continuous acetate enrichment approaches.

To enrich syntrophic acetate-oxidizing bacteria (SAOB), duplicate chemostats were inoculated with sludge from syntrophic acetate oxidation (SAO)-dominated systems and continuously supplied with acetate (0.4 or 7.5 g l-1 ) at high-ammonia levels. The chemostats were operated under mesophilic (37°C) or thermophilic (52°C) temperature for about six hydraulic retention times (HRT 28 days) and were sampled over time. Irrespective of temperature, a methane content of 64-69% and effluent acetate level of 0.4-1.0 g l-1 were recorded in chemostats fed high acetate. Low methane production in the low-acetate chemostats indicated that the substrate supply was below the threshold for methanization of acetate via SAO. Novel representatives within the family Clostridiales and genus Syntrophaceticus (class Clostridia) were identified to represent putative SAOB candidates in mesophilic and thermophilic conditions respectively. Known SAOB persisted at low relative abundance in all chemostats. The hydrogenotrophic methanogens Methanoculleus bourgensis (mesophilic) and Methanothermobacter thermautotrophicus (thermophilic) dominated archaeal communities in the high-acetate chemostats. In line with the restricted methane production in the low-acetate chemostats, methanogens persisted at considerably lower abundance in these chemostats. These findings strongly indicate involvement in SAO and tolerance to high ammonia levels of the species identified here, and have implications for understanding community function in stressed anaerobic processes.

Microbial community dynamics linked to enhanced substrate availability and biogas production of electrokinetically pre-treated waste activated sludge.

The restricted hydrolytic degradation rate of complex organic matter presents a considerable challenge in anaerobic digestion of waste activated sludge (WAS). Within this context, application of pre-treatment of digester substrate has potential for improved waste management and enhanced biogas production. Anaerobic degradation of untreated or electrokinetically pre-treated WAS was performed in two pilot-scale digesters for 132days. WAS electrokinetically pre-treated with energy input 0.066kJ/kg sludge was used in a first phase of operation and WAS pre-treated with energy input 0.091kJ/kg sludge was used in a second phase (each phase lasted at least three hydraulic retention times). Substrate characteristics before and after pre-treatment and effects on biogas digester performance were comprehensively analysed. To gain insights into influences of altered substrate characteristics on microbial communities, the dynamics within the bacterial and archaeal communities in the two digesters were investigated using 16S rRNA gene sequencing (pyrosequencing) and quantitative PCR (qPCR). Specific primers targeting dominant operation taxonomic units (OTUs) and members of the candidate phylum Cloacimonetes were designed to further evaluate their abundance and dynamics in the digesters. Electrokinetic pre-treatment significantly improved chemical oxygen demand (COD) and carbohydrate solubility and increased biogas production by 10-11% compared with untreated sludge. Compositional similarity of the bacterial community during initial operation and diversification during later operation indicated gradual adaptation of the community to the higher solubility of organic material in the pre-treated substrate. Further analyses revealed positive correlations between gene abundance of dominant OTUs related to Clostridia and Cloacimonetes and increased substrate availability and biogas production. Among the methanogens, the genus Methanosaeta dominated in both digesters. Overall, the results showed that electrokinetic pre-treatment of WAS increases substrate solubility and biogas production. Changes in bacterial community composition and abundances of dominant bacterial OTUs were observed during anaerobic degradation of pre-treated WAS, whereas the relative abundance of methanogenic community members remained stable.

Bacterial community composition and fhs profiles of low- and high-ammonia biogas digesters reveal novel syntrophic acetate-oxidising bacteria.

BACKGROUND: Syntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO. RESULTS: All methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO. CONCLUSIONS: The phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.

Microwave and ultrasound pre-treatments influence microbial community structure and digester performance in anaerobic digestion of waste activated sludge.

Comparative analyses of bacterial and archaeal community structures and dynamics in three biogas digesters during start-up and subsequent operation using microwaved, ultrasonicated or untreated waste activated sludge were performed based on 454 pyrosequencing datasets of part of 16S ribosomal RNA sequences and quantitative PCR. The pre-treatment increased the solubility, and thus the availability of the substrate for microbial degradation and significantly affected the succession of the anaerobic community structure over the course of the digestion. Bacteroidetes, Proteobacteria and Firmicutes were the dominant phyla in all digesters throughout operation. Proteobacteria decreased in relative abundance from 23-26 % to 11-13 % in association with enhanced substrate availability. Negative correlations between relative abundance of Alpha-, Beta- and Gammaproteobacteria and the substrate availability and/or biogas production were disclosed in statistical analyses. Clostridiales was the dominant order in Firmicutes, and Clostridiales, Clostridia and Firmicutes relative abundance and richness were shown to positively correlate with substrate availability and biogas generation. Methanogenic communities had a fairly restricted structure, highly dominated by Methanosaeta and Methanobrevibacter phylotypes. A gradual decline in Methanobrevibacter and increased representation of Methanosaeta concilii over time were particularly apparent in the digester receiving untreated waste activated sludge, whereas more diversified archaeal communities were maintained in the pre-treatment digesters. The quantitative PCR analyses revealed a methanogenic community distribution that coincided with the 454 pyrosequencing data.

Ammonia threshold for inhibition of anaerobic digestion of thin stillage and the importance of organic loading rate.

Biogas production from nitrogen-rich feedstock results in release of ammonia (NH3), causing inhibition of the microbial process. The reported threshold ammonia value for stable biogas production varies greatly between studies, probably because of differences in operating conditions. Moreover, it is often difficult to separate the effect of ammonia inhibition from that of organic loading rate (OLR), as these two factors are often interrelated. This study attempted to distinguish the effects of ammonia and OLR by analysis of two laboratory-scale biogas reactors operating with thin stillage and subjected to an increase in free ammonia (from 0.30 to 1.1 g L(-1)) either by addition of an external nitrogen source (urea) or by increasing the OLR (3.2-6.0 g volatile solids L(-1) d(-1)). The results showed that ammonia concentration was detrimental for process performance, with the threshold for stability in both processes identified as being about 1 g NH3-N L(-1), irrespective of OLR. Analysis of the methanogenic community showed limited differences between the two reactors on order level and a clear increase in the abundance of Methanomicrobiales, particularly Methanoculleus sp., in response to increasing ammonia concentration. Further comprehensive molecular analysis revealed that diverse Methanoculleus species dominated in the reactors at a given ammonia level at different OLR. The acetogenic community was clearly affected by both ammonia concentration and OLR, suggesting that the volatile fatty acid load in relation to the higher OLR was important for the dynamics of this community.