Evaluation of process performance and retained sludge properties of a psychrophilic UASB reactor for treatment of iso-plophyl alcohol (2-propanol)-containing wastewater.

In this study, a continuous feeding experiment was conducted with synthetic iso-plophyl alcohol (2-propanol)-containing wastewater using a lab-scale psychrophilic UASB reactor to evaluate process performance and retained sludge properties. For smooth acclimation, methanogenic granular sludge was seeded and a proportion of 2-propanol in the synthetic wastewater containing sucrose and volatile fatty acids was increased stepwise from 0% to 30%, 60% and then 90% of COD (chemical oxygen demand). As a result, after a 4-week period for acclimation to 2-propanol degradation, a COD removal rate of 95% was achieved at an organic loading rate (OLR) of 8.4 kg COD/m3/day. Additionally, the physical properties of the retained granular sludge were maintained even when the reactor was supplied with 2-propanol-rich wastewater for more than 200 days. From the batch assays using serum bottles, methanogenic degradation of 2-propanol was observed with acetone accumulation. By comparison, 2-propanol degradation was clearly inhibited in the presence of chloroform as a specific inhibitor of methanogen. A domain archaeal community structure analysis targeting 16S rRNA genes showed the relative abundance of the genus Methanospillium was increased in the 2-propanol acclimated sludge. These results suggested Methanospillium related species in the granular sludge appreciably contributed to the direct degradation of 2-proapanol into acetone under an anaerobic condition.

Methanospirillum psychrodurum sp. nov., isolated from wetland soil.

A psychrotolerant methanogenic strain, X-18(T), was isolated from the soil of the Madoi wetland at Qinghai, Tibetan plateau, China. Cells were wavy rods (11-62 µm long) with blunt tapered ends and Gram-stain-negative. Strain X-18(T) grew strictly anaerobically and produced methane exclusively from H2/CO2. Growth occurred in the temperature range of 4-32 °C and optimally at 25 °C. Growth pH ranged from 6.5 to 8.0 and the optimum was 7.0. The G+C content of the genomic DNA of strain X-18(T) was 44.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences and the alpha subunit of methyl-coenzyme M reductase indicated that strain X-18(T) was affiliated to the genus Methanospirillum and was most closely related to Methanospirillum lacunae Ki8-1(T), with 96.3% 16S rRNA gene sequence similarity. However, strain X-18(T) could be distinguished from the existing species of the genus Methanospirillum by its lower growth temperature and obligate hydrogenotrophic methanogenesis. On the basis of phenotypic characteristics and phylogenetic analysis, strain X-18(T) represents a novel species of the genus Methanospirillum, for which the name Methanospirillum psychrodurum sp. nov. is proposed and strain X-18(T) is assigned as the type strain ( = CGMCC 1.5186(T) = JCM 19216(T)).

Methanospirillum stamsii sp. nov., a psychrotolerant, hydrogenotrophic, methanogenic archaeon isolated from an anaerobic expanded granular sludge bed bioreactor operated at low temperature.

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)).

Psychrophilic anaerobic membrane bioreactor treatment of domestic wastewater.

A bench-scale anaerobic membrane bioreactor (AnMBR) equipped with submerged flat-sheet microfiltration membranes was operated at psychrophilic temperature (15 °C) treating simulated and actual domestic wastewater (DWW). Chemical oxygen demand (COD) removal during simulated DWW operation averaged 92 ± 5% corresponding to an average permeate COD of 36 ± 21 mg/L. Dissolved methane in the permeate stream represented a substantial fraction (40-50%) of the total methane generated by the system due to methane solubility at psychrophilic temperatures and oversaturation relative to Henry's law. During actual DWW operation, COD removal averaged 69 ± 10%. The permeate COD and 5-day biochemical oxygen demand (BOD(5)) averaged 76 ± 10 mg/L and 24 ± 3 mg/L, respectively, indicating compliance with the U.S. EPA's standard for secondary effluent (30 mg/L BOD(5)). Membrane fouling was managed using biogas sparging and permeate backflushing and a flux greater than 7 LMH was maintained for 30 days. Comparative fouling experiments suggested that the combination of the two fouling control measures was more effective than either fouling prevention method alone. A UniFrac based comparison of bacterial and archaeal microbial communities in the AnMBR and three different inocula using pyrosequencing targeting 16S rRNA genes suggested that mesophilic inocula are suitable for seeding psychrophilic AnMBRs treating low strength wastewater. Overall, the research described relatively stable COD removal, acceptable flux, and the ability to seed a psychrophilic AnMBR with mesophilic inocula, indicating future potential for the technology in practice, particularly in cold and temperate climates where DWW temperatures are low during part of the year.

Methanospirillum lacunae sp. nov., a methane-producing archaeon isolated from a puddly soil, and emended descriptions of the genus Methanospirillum and Methanospirillum hungatei.

A mesophilic, hydrogenotrophic methanogen, designated strain Ki8-1(T), was isolated from soil. Cells were strictly anaerobic, Gram-stain-negative, non-sporulating, motile by means of a single flagellum or tufted flagella, and curved or wavy rod-shaped (11-25 µm long). The temperature and pH for optimum growth were 30 °C and 7.5. The strain grew best in basal medium without the addition of NaCl. Methane was produced from H(2) and formate. Acetate or yeast extract was required for growth. The G+C content of the genomic DNA of strain Ki8-1(T) was 45.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Ki8-1(T) was a member of the genus Methanospirillum and showed 95.1% sequence similarity to Methanospirillum hungatei NBRC 100397(T). On the basis of its phenotypic characteristics and phylogenetic position, strain Ki8-1(T) is considered to represent a novel species of the genus Methanospirillum, for which the name Methanospirillum lacunae sp. nov. is proposed. The type strain is Ki8-1(T) (NBRC 104920(T) =JCM 16384(T) =DSM 22751(T)). Emended descriptions of the genus Methanospirillum and of Methanospirillum hungatei are also provided.

Changes in process performance and microbial characteristics of retained sludge during low-temperature operation of an EGSB reactor.

A lab-scale expanded granular sludge bed (EGSB) reactor was seeded with granular sludge and operated to investigate the influence of temperature decrease on both process performance and the microbial community structure of the granular sludge. Synthetic wastewater containing sucrose and volatile fatty acids was used as feed. The EGSB reactor was brought online at a starting temperature of 15 degrees C and was reduced stepwise to a final temperature of 5 degrees C. The reactor exhibited sufficient COD removal efficiency between 10 degrees C and 15 degrees C. However at 5 degrees C serious deterioration of process performance was observed. The methane-producing activity of the retained sludge increased when it was compared to the activity of the seed sludge (day 0) during 10 degrees C to 15 degrees C operation. When hydrogen fed, sludge showed much higher methanogenic activity as compared with seed sludge activity at test temperatures of 15 degrees C and 20 degrees C on day 196 of reactor operation. At this time, proliferation of the genus Methanospirillum in the retained sludge was observed and a decrease in Methanobacterium species was also measured. Throughout the experiment, the genus Methanosaeta was detected in abundance and the community structure of the Domain Bacteria was stably maintained. The sugar-degrading acid-forming bacteria, Lactococcus and Anaerovibrio were detected in the retained sludge throughout the experiment as well and the propionate-degrading acetogen Syntrophobacter fumaroxidans was also detected, although its population size decreased at 5 degrees C.

Characterization of the community structure of a dechlorinating mixed culture and comparisons of gene expression in planktonic and biofloc-associated "Dehalococcoides" and Methanospirillum species.

This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming "Dehalococcoides ethenogenes" strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of "Dehalococcoides" and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan.

A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H(2)-CO(2) at 20 degrees C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H(2)-CO(2), 2-propanol-CO(2), or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field.