Yan-Ling Qiu

Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments

ABSTRACT The classical perception of members of the gram-positive Desulfotomaculum cluster I as sulfate-reducing bacteria was recently challenged by the isolation of new representatives lacking the ability for anaerobic sulfate respiration. For example, the two described syntrophic propionate-oxidizing species of the genus Pelotomaculum form the novel Desulfotomaculum subcluster Ih. In the present study, we applied a polyphasic approach by using cultivation-independent and culturing techniques in order to further characterize the occurrence, abundance, and physiological properties of subcluster Ih bacteria in low-sulfate, methanogenic environments. 16S rRNA (gene)-based cloning, quantitative fluorescence in situ hybridization, and real-time PCR analyses showed that the subcluster Ih population composed a considerable part of the Desulfotomaculum cluster I community in almost all samples examined. Additionally, five propionate-degrading syntrophic enrichments of subcluster Ih bacteria were successfully established, from one of which the new strain MGP was isolated in coculture with a hydrogenotrophic methanogen. None of the cultures analyzed, including previously described Pelotomaculum species and strain MGP, consumed sulfite, sulfate, or organosulfonates. In accordance with these phenotypic observations, a PCR-based screening for dsrAB (key genes of the sulfate respiration pathway encoding the alpha and beta subunits of the dissimilatory sulfite reductase) of all enrichments/(co)cultures was negative with one exception. Surprisingly, strain MGP contained dsrAB, which were transcribed in the presence and absence of sulfate. Based on these and previous findings, we hypothesize that members of Desulfotomaculum subcluster Ih have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction.

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the First Cultured Anaerobe Capable of Degrading Phenol to Acetate in Obligate Syntrophic Associations with a Hydrogenotrophic Methanogen

ABSTRACT Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UIT, was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H2-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 μM) and benzoate (a maximum of 11 μM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 μM) and benzoate (maximum, 92 μM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UIT in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UIT belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UIT, and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.

Effect of inoculum/substrate ratio on methane yield and orthophosphate release from anaerobic digestion of Microcystis spp.

A batch anaerobic test was conducted to evaluate the effects of inoculum/substrate ratios (ISRs) on the methane yield and orthophosphate release from the anaerobic digestion of Microcystis spp. The results demonstrated an obvious influence on methane yield and orthophosphate release by ISR. The maximum methane yield decreased from 140.48 to 94.42 mL/gVS when the ISR decreased from 2.0 to 0.5. The highest maximum methane yield calculated from Ørskov equation was 153.66 mL/gVS at ISR value of 1.0. The values of pH, ammonia and volatile fatty acids (VFAs) corroborated the appropriate stability of this anaerobic process.

Biogas production and microcystin biodegradation in anaerobic digestion of blue algae

In order to demonstrate the potentials of both bioenergy production and microcystin (MC) biodegradation in methanogenic conditions, the biogas production, process stability and the variation of MC concentration in anaerobic digestion of blue algae from Taihu Lake were studied. A methane yield of 189.89 mL g−1 VS was obtained from the digester, and the average methane concentration in the biogas was 36.72%. During the digestion, the pH value was fairly constant (6.8 to 7.6), and soluble chemical oxygen demand (SCOD) kept at a relatively stable level. The concentration of total volatile fatty acid (VFA) increased significantly in the first 8 days, and then decreased, showing no inhibition on the digestion. The results also showed that the concentration of MC could be significantly reduced from 1220.19 μg L−1 to 35.17 μg L−1 during the methanogenic process, which followed the first order kinetics well. These findings suggest that anaerobic digestion of blue algae can both generate renewable energy biogas and lead to biodegradation of MC, presenting significant potentials for bioremediation of MC and post-treatment of blue algae.

Acetobacteroides hydrogenigenes gen. nov., sp. nov., an anaerobic hydrogen-producing bacterium in the family Rikenellaceae isolated from a reed swamp.

A strictly anaerobic, mesophilic, carbohydrate-fermenting, hydrogen-producing bacterium, designated strain RL-C(T), was isolated from a reed swamp in China. Cells were Gram-stain-negative, catalase-negative, non-spore-forming, non-motile rods measuring 0.7-1.0 µm in width and 3.0-8.0 µm in length. The optimum temperature for growth of strain RL-C(T) was 37 °C (range 25-40 °C) and pH 7.0-7.5 (range pH 5.7-8.0). The strain could grow fermentatively on yeast extract, tryptone, arabinose, glucose, galactose, mannose, maltose, lactose, glycogen, pectin and starch. The main end products of glucose fermentation were acetate, H2 and CO2. Organic acids, alcohols and amino acids were not utilized for growth. Yeast extract was not required for growth; however, it stimulated growth slightly. Nitrate, sulfate, sulfite, thiosulfate, elemental sulfur and Fe(III) nitrilotriacetate were not reduced as terminal electron acceptors. Aesculin was hydrolysed but not gelatin. Indole and H2S were produced from yeast extract. The G+C content of the genomic DNA was 51.2 mol%. The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. The most abundant polar lipid of strain RL-C(T) was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that the isolate belongs to the uncultured Blvii28 wastewater-sludge group (http://www.arb-silva.de/) in the family Rikenellaceae of the phylum Bacteroidetes, and shared low sequence similarities with the related species Alistipes shahii WAL 8301(T) (81.8 %), Rikenella microfusus ATCC 29728(T) (81.7 %) and Anaerocella delicata WN081(T) (80.9 %). On the basis of these data, a novel species in a new genus of the family Rikenellaceae is proposed, Acetobacteroides hydrogenigenes gen. nov., sp. nov. The type strain of the type species is RL-C(T) ( = JCM 17603(T) = DSM 24657(T) = CGMCC 1.5173(T)).

Molecular characterization of bacterial and archaeal communities in a full-scale anaerobic reactor treating corn straw

A 16S rRNA gene-based method was used to characterize the structure of bacterial and archaeal communities in a full-scale, anaerobic reactor treating corn straw. Degradability experiment indicated biogas slurry had high microbial activity, the TS removal rate was 53% and the specific methanogenic activity was 86 mL CH4 g VSS(-1) d(-1). During anaerobic degradation of corn straw, volatile acids and aromatic compounds (p-cresol, phenylpropionate, phenol and benzoate) were detected as transient intermediates. Phylogenetic analysis revealed bacterial community exhibited high diversity, 69 bacterial phylotypes in 13 phyla were identified. Firmicutes (48.3%), Chloroflexi (20.1%), Actinobacteria (9.1%), Bacteroidetes (7.7%), and Proteobacteria (7.2%) represented the most abundant bacterial phyla. Hydrolytic and fermentative bacteria were major bacterial populations. Moreover, a relatively high proportion of syntrophic propionate and aromatic compounds degrading bacteria were detected. In the archaeal clone library, 11 archaeal phylotypes affiliated with two phyla of Crenarchaeota (10%) and Euryarchaeota (90%) were identified.

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.

Bioaugmentation with an acetate-type fermentation bacterium Acetobacteroides hydrogenigenes improves methane production from corn straw.

The effect of bioaugmentation with an acetate-type fermentation bacterium in the phylum Bacteroidetes on the anaerobic digestion of corn straw was evaluated by batch experiments. Acetobacteroides hydrogenigenes is a promising strain for bioaugmentation with relatively high growth rate, hydrogen yields and acetate tolerance, which ferments a broad spectrum of pentoses, hexoses and polyoses mainly into acetate and hydrogen. During corn straw digestion, bioaugmentation with A. hydrogenigenes led to 19-23% increase of the methane yield, with maximum of 258.1 mL/g-corn straw achieved by 10% inoculation (control, 209.3 mL/g-corn straw). Analysis of lignocellulosic composition indicated that A. hydrogenigenes could increase removal rates of cellulose and hemicelluloses in corn straw residue by 12% and 5%, respectively. Further experiment verified that the addition of A. hydrogenigenes could improve the methane yields of methyl cellulose and xylan (models for cellulose and hemicelluloses, respectively) by 16.8% and 7.0%.

Bioaugmentation of Hydrogenispora ethanolica LX-B affects hydrogen production through altering indigenous bacterial community structure.

Bioaugmentation can facilitate hydrogen production from complex organic substrates, but it still is unknown how indigenous microbial communities respond to the added bacteria. Here, using a Hydrogenispora ethanolica LX-B (named as LX-B) bioaugmentation experiments, the distribution of metabolites and the responses of indigenous bacterial communities were investigated via batch cultivation (BC) and repeated batch cultivation (RBC). In BC the LX-B/sludge ratio of 0.12 achieved substantial high hydrogen yield, which was over twice that of control. In RBC one-time bioaugmentation and repeated batch bioaugmentation of LX-B resulted in the hydrogen yield that was average 1.2-fold and 0.8-fold higher than that in control, respectively. This improved hydrogen production performance mainly benefited from a shift in composition of the indigenous bacterial community caused by LX-B bioaugmentation. The findings represented an important step in understanding the relationship between bioaugmentation, a shift in bacterial communities, and altered bioreactor performance.

Hydrogenispora ethanolica gen. nov., sp nov., an anaerobic carbohydrate-fermenting bacterium from anaerobic sludge

An anaerobic, spore-forming, ethanol-hydrogen-coproducing bacterium, designated LX-BT, was isolated from an anaerobic sludge treating herbicide wastewater. Cells of strain LX-BT were non-motile rods (0.3-0.5×3.0-18.0 µm). Spores were terminal with a bulged sporangium. Growth occurred at 20-50 °C (optimum 37-45 °C), pH 5.0-8.0 (optimum pH 6.0-7.7) and 0-2.5% (w/v) NaCl. The strain could grow fermentatively on glucose, maltose, arabinose, fructose, xylose, ribose, galactose, mannose, raffinose, sucrose, pectin, starch, glycerol, fumarate, tryptone and yeast extract. The major end-products of glucose fermentation were acetate, ethanol and hydrogen. Yeast extract was not required but stimulated growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, anthraquinone-2,6-disulfonate, fumarate and Fe (III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of the genomic DNA was 56.1 mol%. The major cellular fatty acids were anteiso-C15:0, iso-C14:0 and C16:0. The most abundant polar lipids of strain LX-BT were diphosphatidylglycerol and phosphatidylglycerol. 16S rRNA gene sequence analysis revealed that it belongs to an as-yet-unidentified taxon at the order- or class-level (OPB54) within the phylum Firmicutes, showing 86.5% sequence similarity to previously described species of the Desulfotomaculum cluster. The name Hydrogenispora ethanolica gen. nov., sp. nov. is proposed to accommodate strain LX-BT (=DSM 25471T=JCM 18117T=CGMCC 1.5175T) as the type strain.