Molecular microbial ecology of stable versus failing rice straw anaerobic digesters

Waste rice straw (RS) is generated in massive quantities around the world and is often burned, creating greenhouse gas and air quality problems. Anaerobic digestion (AD) may be a better option for RS management, but RS is presumed to be comparatively refractory under anaerobic conditions without pre-treatment or co-substrates. However, this presumption assumes frequent reactor feeding regimes but less frequent feeding may be better for RS due to slow hydrolysis rates. Here, we assess how feeding frequency (FF) and organic loading rate (OLR) impacts microbial communities and biogas production in RS AD reactors. Using 16S rDNA amplicon sequencing and bioinformatics, microbial communities from five bench-scale bioreactors were characterized. At low OLR (1.0 g VS l⁻¹ day⁻¹), infrequently fed units (once every 21 days) had higher specific biogas yields than more frequent feeding (five in 7 days), although microbial community diversities were statistically similar (P > 0.05; ANOVA with Tukey comparison). In contrast, an increase in OLR to 2.0 g VS l⁻¹ day⁻¹ significantly changed Archaeal and fermenting Eubacterial sub-communities and the least frequency fed reactors failed. ‘Stable’ reactors were dominated by Methanobacterium, Methanosarcina and diverse Bacteroidetes, whereas ‘failed’ reactors saw shifts towards Clostridia and Christensenellaceae among fermenters and reduced methanogen abundances. Overall, OLR impacted RS AD microbial communities more than FF. However, combining infrequent feeding and lower OLRs may be better for RS AD because of higher specific yields.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

Effect of Seawater–Sewage Cross-Transplants on Bacterial Metabolism and Diversity

Bioassays experiments were conducted to determine the metabolic and community composition response of bacteria to transplants between relatively pristine coastal seawater and sewage-impacted seawater. There were four treatments: (1) pristine seawater bacteria?+?pristine seawater (Pb?+?Pw), (2) sewage-impacted bacteria?+?sewage-impacted water (Sb?+?Sw), (3) pristine seawater bacteria?+?sewage-impacted water (Pb?+?Sw), and (4) sewage-impacted bacteria?+?pristine seawater (Sb?+?Pw). Sewage-derived DOC was more labile and readily utilized by bacteria, which favored the growth of high nucleic acid (HNA) bacteria, resulting in high bacterial production (BP, 113?±?4.92 to 130?±?15.8 μg C l^?1?day^?1) and low respiration rate (BR, <67?±?11.3 μg C l^?1?day^?1), as well as high bacterial growth efficiency (BGE, 0.68?±?0.09 to 0.71?±?0.05). In contrast, at the relatively pristine site, bacteria utilized natural marine-derived dissolved organic matter (DOM) at the expense of lowering their growth efficiency (BGE, <0.32?±?0.02) with low BP (<62?±?6.3 μg C l^?1?day^?1) and high BR 133?±?14.2 μg C l^?1?day^?1). Sewage DOM input appeared to alter the partitioning of carbon between respiration and production of bacteria, resulting in a shift toward higher BGE, which would not enhance oxygen consumption. Taxonomic classification based on 454 pyrosequencing reads of the 16S rRNA gene amplicons revealed that changes in bacterial community structure occurred when seawater bacteria were transferred to the eutrophic sewage-impacted water. Sewage DOM fueled the growth of Gammma-proteobacteria and Epsilson-proteobacteria and reduced the bacterial richness, but the changes in the community were not apparent when sewage-impacted bacteria were transferred to pristine seawater.     

Hydrolysis,acidification and methanogenesis during low-temperature anaerobic digestion of dilute dairy wastewater in an inverted fluidised bioreactor

The application of low-temperature (10 °C) anaerobic digestion (LtAD) for the treatment of complex dairy-based wastewater in an inverted fluidised bed (IFB) reactor was investigated. Inadequate mixing intensity provoked poor hydrolysis of the substrate (mostly protein), which resulted in low chemical oxygen demand (COD) removal efficiency throughout the trial, averaging ~69 % at the best operational period. Overgrowth of the attached biomass to the support particles (Extendospheres) induced bed stratification by provoking agglutination of the particles and supporting their washout by sedimentation, which contributed to unstable bioprocess performance at the organic loading rates (OLRs) between 0.5 and 5 kg COD m^?3 day^?1. An applied OLR above 2 kg COD m^?3 day^?1 additionally promoted acidification and strongly influenced the microbial composition and dynamics. Hydrogenotrophic methanogens appeared to be the mostly affected group by the Extendospheres particle washout as a decrease in their abundance was observed by quantitative PCR analysis towards the end of the trial, although the specific methanogenic activity and maximum substrate utilisation rate on H₂/CO₂ indicated high metabolic activity and preference towards hydrogenotrophic methanogenesis of the reactor biomass at this stage. The bacterial community in the bioreactor monitored via denaturing gradient gel electrophoresis (DGGE) also suggested an influence of OLR stress on bacterial community structure and population dynamics. The data presented in this work can provide useful information in future optimisation of fluidised reactors intended for digestion of complex industrial wastewaters during LtAD.     

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.     

Evaluation of an electronic nose for the early detection of organic overload of anaerobic digesters

This study aimed at analysing the utilization of an electronic nose (e-nose) to serve as a specific monitoring tool for anaerobic digestion process, especially for detecting organic overload. An array of non specific metal oxide semiconductor gas sensors were used to detect process faults due to organic overload events in twelve 1.8-L anaerobic semi-continuous reactors. Three different load strategies were followed: (1) a cautious organic load (1.3 gVS L^?1 day^?1); (2) an increasing load strategy (1.3–5.3 gVS L^?1 day^?1) and (3) a cautious organic load with load pulses of up to 12 gVS L^?1 day^?1. A first monitoring campaign was conducted with three different substrates: sucrose, maize oil and a mix of sucrose/oil during 60 days. The second campaign was run with dry sugar beet pulp for 45 days. Hotelling’s T ² value and upper control limit to a reference set of digesters fed with a cautious OLR (1.3 gVS L^?1 day^?1) was used as indirect state variable of the reactors. Overload situations were identified by the e-nose apparatus with Hotelling’s T ² values at least four times higher in magnitude than the upper control limit of 23.7. These results confirmed that the e-nose technology appeared promising for online detection of process imbalances in the domain of anaerobic digestion.     

Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate

The bioconversion of renewable raw material to biogas by anaerobic microbial fermentation processes in completely stirred tank reactors (CSTR) is a valuable alternative resource of energy especially for rural areas. However, knowledge about the microorganisms involved in the degradation of plant biomass is still poor. In this study, a first analysis of the biogas-forming process within a CSTR fed continuously with fodder beet silage as mono-substrate is presented in the context of molecular data on the microbial community composition. As indicated by the conventional process parameters like pH value, content of volatile fatty acids, N:P ratio and the biogas yield, the biogas-forming process within the CSTR occurred with a stable and efficient performance. The average biogas yield based on volatile solids was 0.87m(3)kg(-1) at an organic loading rate of 1.2-2.3kgm(-3)d(-1). This amounts to 94% of the theoretical maximum. In order to identify microorganisms within the CSTR, a 16S rDNA clone library was constructed by PCR amplification applying a prokaryote-specific primer set. One hundred and forty seven clones were obtained and subsequently characterized by amplified rDNA restriction analysis (ARDRA). The sequences of 60 unique ARDRA patterns were estimated in a length of approximately 800-900bp each. Four of them were assigned to the domain Archaea and 56 to the domain Bacteria. Within the domain Archaea, all clones showed a close relationship to methanogenic species. Major bacterial groups represented in the clone library were the class Clostridia of the phylum Firmicutes (22% of all 16S rDNA clones), the class Deltaproteobacteria of the phylum Proteobacteria (24%), the class Bacilli of the phylum Firmicutes (22%) and members of the phylum Bacteroidetes (21%). Within these major groups, the highest biodiversity was found within the class Clostridia (35% of all operational taxonomic units). Members of the phyla Actinobacteria and Spirochaetes were represented only by 5 and 2 clonal sequences, respectively.     

Impact of increasing NaCl concentrations on the performance and community composition of two anaerobic reactors

The anaerobic treatment of saline effluents using halophilic and halotolerant microbial consortia is of major interest. Inhibition of anaerobic digestion is known to occur at high salt content. However, it seems that the suitable adaptation of an anaerobic sludge makes possible the treatment of saline wastewater. In this study, a non-saline anaerobic sludge was inoculated in two anaerobic batch reactors operating with a different substrate (distillery vinasse and ethanol) and subjected to increasing NaCl concentrations. The performance of the digesters appeared to be highly dependent on the nature of the substrate, and a similar level of inhibition (i.e. around 90% of the specific loading rate and specific methanogenic activity) was stated at 10 g l⁻¹ of NaCl with distillery vinasse and 60 g l⁻¹ of NaCl with ethanol. The characterization of the microflora and its adaptation to increasing NaCl conditions were also investigated using molecular tools based on the analysis of genomic 16S rDNA. The microbial communities revealed a high diversity that could be maintained in both reactors despite the increase in NaCl concentrations.     

Methanogenesis,sulfate reduction and crude oil biodegradation in hot Alaskan oilfields

Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6–807 µmol S l^?1 day^?1), but of lesser consequence in Field B (0.1–10 µmol S l^?1 day^?1). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular‐based microbial community analysis of the methanogenic oil‐utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H₂‐using family Methanobacteriaceae. The findings demonstrate that oilfield‐associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Methane production potential and methanogenic archaea community dynamics along the Spartina alterniflora invasion chronosequence in a coastal salt marsh

Invasion by the exotic species Spartina alterniflora, which has high net primary productivity and superior reproductive capacity compared with native plants, has led to rapid organic carbon accumulation and increased methane (CH₄) emission in the coastal salt marsh of China. To elucidate the mechanisms underlying this effect, the methanogen community structure and CH₄ production potential as well as soil organic carbon (SOC), dissolved organic carbon, dissolved organic acids, methylated amines, aboveground biomass, and litter mass were measured during the invasion chronosequence (0–16 years). The CH₄ production potential in the S. alterniflora marsh (range, 2.94–3.95 μg kg^?1 day^?1) was significantly higher than that in the bare tidal mudflat. CH₄ production potential correlated significantly with SOC, acetate, and trimethylamine concentrations in the 0–20 cm soil layer. The abundance of methanogenic archaea also correlated significantly with SOC, and the dominant species clearly varied with S. alterniflora-driven SOC accumulation. The acetotrophic Methanosaetaceae family members comprised a substantial proportion of the methanogenic archaea in the bare tidal mudflat while Methanosarcinaceae family members utilized methylated amines as substrates in the S. alterniflora marsh. Ordination analysis indicated that trimethylamine concentration was the primary factor inducing the shift in the methanogenic archaea composition, and regressive analysis indicated that the facultative family Methanosarcinaceae increased linearly with trimethylamine concentration in the increasingly sulfate-rich salt marsh. Our results indicate that increased CH₄ production during the S. alterniflora invasion chronosequence was due to increased levels of the non-competitive substrate trimethylamine and a shift in the methanogenic archaea community.     

A quantitative study of eicosapentaenoic acid (EPA) production by Nannochloropsis gaditana for aquaculture as a function of dilution rate,temperature and average irradiance

Different pilot-scale outdoor photobioreactors using medium recycling were operated in a greenhouse under different environmental conditions and the growth rates (0.1 to 0.5 day^?1) obtained evaluated in order to compare them with traditional systems used in aquaculture. The annualized volumetric growth rate for Nannochloropsis gaditana was 0.26 g l^?1 day^?1 (peak 0.4 g l^?1 day^?1) at 0.4 day^?1 in a 5-cm wide flat-panel bioreactor (FP-PBR). The biomass productivity achieved in this reactor was 10-fold higher than in traditional reactors, reaching values of 28 % and 45 % dry weight (d.w.) of lipids and proteins, respectively, with a 4.3 % (d.w.) content of eicosapentaenoic acid (EPA). A model for predicting EPA productivity from N. gaditana cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effect of temperature and average irradiance on EPA content is also studied. The maximum EPA productivity attained is 30 mg l^?1 day^?1.     

Arthrospira platensis biomass with high protein content cultivated in continuous process using urea as nitrogen source

Aims: Arthrospira platensis has been studied for single‐cell protein production because of its biomass composition and its ability of growing in alternative media. This work evaluated the effects of different dilution rates (D) and urea concentrations (N₀) on A. platensis continuous culture, in terms of growth, kinetic parameters, biomass composition and nitrogen removal. Methods and results: Arthrospira platensis was continuously cultivated in a glass‐made vertical column photobioreactor agitated with Rushton turbines. There were used different dilution rates (0·04–0·44 day^?1) and urea concentrations (0·5 and 5 mmol l^?1). With N₀ = 5 mmol l^?1, the maximum steady‐state biomass concentration was1415 mg l^?1, achieved with D = 0·04 day^?1, but the highest protein content (71·9%) was obtained by applying D = 0·12 day^?1, attaining a protein productivity of 106·41 mg l^?1 day^?1. Nitrogen removal reached 99% on steady‐state conditions. Conclusions: The best results were achieved by applying N₀ = 5 mmol l^?1; however, urea led to inhibitory conditions at D ≥ 0·16 day^?1, inducing the system wash‐out. The agitation afforded satisfactory mixture and did not harm the trichomes structure. Significance and Impact of the Study: These results can enhance the basis for the continuous removal of nitrogenous wastewater pollutants using cyanobacteria, with an easily assembled photobioreactor.     

Microbiological investigations of high-temperature horizons of the Kongdian petroleum reservoir in connection with field trial of a biotechnology for enhancement of oil recovery

The physicochemical conditions and microbiological characteristics of the formation waters of the Kongdian oilfield of the Dagang oilfield (China) were studied. It was demonstrated that this oilfield is a high-temperature ecosystem with formation waters characterized by low mineralization. The concentrations of nitrogen and phosphorus compounds, as well as of electron acceptors, are low. Oil and oil gas are the main organic matter sources. The oilfield is exploited with water-flooding. The oil stratum was inhabited mostly by anaerobic thermophilic microorganisms, including fermentative (10²–10⁵ cells/ml), sulfate-reducing (0–10² cells/ml), and methanogenic (0–10³ cells/ml) microorganisms. Aerobic bacteria were detected mainly in the near-bottom zone of injection wells. The rate of sulfate reduction varied from 0.002 to 18.940 μg S^2? l^?1 day^?1 and the rate of methanogenesis from 0.012 to 16.235 μg CH₄ l^?1 day^?1. Microorganisms with great biotechnological potential inhabited the oilfield. Aerobic thermophilic bacteria were capable of oxidizing oil with formation of biomass, the products of partial oxidation of oil (volatile acids), and surfactants. During growth on the culture liquid of oil-oxidizing bacteria, methanogenic communities produced methane and carbon dioxide, which also had oil-releasing capabilities. Using various labeled tracers, the primary filtration flows of injected solutions at the test site were studied. Our comprehensive investigations allowed us to conclude that the method for microbial enhancement of oil recovery based on the activation of the stratal microflora can be applied in the Kongdian oilfield.     

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil

Temperature is an important factor controlling CH(4) production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37 degrees C or in a temperature gradient block covering the same temperature range at intervals of 1 degrees C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH(4) production rates increased with temperature, with an apparent activation energy of 61 kJ mol(-1). Steady-state partial pressures of the methanogenic precursor H(2) also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H(2) plus CO(2)-dependent methanogenesis was kept at -20 to -25 kJ mol of CH(4)(-1) over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 microM. Simultaneously, the relative contribution of H(2) as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to (14)CH(4), so that the carbon and electron flow to CH(4) was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

An Outbreak of Nonflocculating Catabolic Populations Caused the Breakdown of a Phenol-Digesting Activated-Sludge Process

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter⁻¹ day⁻¹ and then to 1.5 g liter⁻¹ day⁻¹. After the loading rate was increased to 1.5 g liter⁻¹ day⁻¹, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter⁻¹ day⁻¹. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter⁻¹ day⁻¹. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members

Microbial conversion of organic waste or harvested plant material into biogas has become an attractive technology for energy production. Biogas is produced in reactors under anaerobic conditions by a consortium of microorganisms which commonly include bacteria of the genus Clostridium. Since the genus Clostridium also harbors some highly pathogenic members in its phylogenetic cluster I, there has been some concern that an unintended growth of such pathogens might occur during the fermentation process. Therefore this study aimed to follow how process parameters affect the diversity of Bacteria in general, and the diversity of Clostridium cluster I members in particular. The development of both communities was followed in model biogas reactors from start-up during stable methanogenic conditions. The biogas reactors were run with either cattle or pig manures as substrates, and both were operated at mesophilic and thermophilic conditions. The structural diversity was analyzed independent of cultivation using a PCR-based detection of 16S rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP). Genetic profiles indicated that both bacterial and clostridial communities evolved in parallel, and the community structures were highly influenced by both substrate and temperature. Sequence analysis of 16S rRNA genes recovered from prominent bands from SSCP profiles representing Clostridia detected no pathogenic species. Thus, this study gave no indication that pathogenic clostridia would be enriched as dominant community members in biogas reactors fed with manure.     

Improving the performance of an aerobic membrane bioreactor (MBR) treating pharmaceutical wastewater with powdered activated carbon (PAC) addition

In this study, the effects of organic loading rate (OLR) and the addition of powdered activated carbon (PAC) on the performance and membrane fouling of MBR were conducted to treat real pharmaceutical process wastewater. Over 145 days of operation, the MBR system was operated at OLRs ranging from 1 to 2 kg COD m^?3 day^?1 without sludge wasting. The addition of PAC provided an improvement in the flux, despite an increase in the OLR:PAC ratio. The results demonstrated that the hybrid PAC-MBR system maintained a reduced amount of membrane fouling and steadily increased the removal performance of etodolac. PAC addition reduced the deposition of extracellular polymeric substance and organic matter on the membrane surface and resulted an increase in COD removal even at higher OLRs with low PAC addition. Membrane fouling mechanisms were investigated using combined adsorption fouling models. Modified fouling index values and normalized mass transfer coefficient values indicated that predominant fouling mechanism was cake adsorption.     

Biomass production and energy source of thermophiles in a Japanese alkaline geothermal pool

Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85°C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day^?1, similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 μg C l^?1 h^?1. This production was 2–75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H₂‐oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H₂ in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H₂ plays an important role as a primary energy source of thermophiles in the geothermal pool.