Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: IV. Adaptation of Activated Sludge to Utilization of Aromatic Compounds 1

An activated sludge from a sewage treatment plant and a laboratory activated sludge developed on an artificial waste were compared for their ability to utilize 11 aromatic compounds. There were several significant differences between them. The laboratory sludge contained higher numbers of organisms and metabolized the aromatics to a greater extent. Laboratory activated sludges acclimated to utilization of the aromatics differed from each other in population structure and the pattern of oxygen consumption with aromatic substrates. The oxidative patterns of uncontrolled mixed populations were unreliable for investigating metabolic pathways. Extracts of the various sludges elevated the plate counts of the sludges.     

Identification of some of the major groups of bacteria in efficient and nonefficient biological phosphorus removal activated sludge systems.

To investigate the bacteria that are important to phosphorus (P) removal in activated sludge, microbial populations were analyzed during the operation of a laboratory-scale reactor with various P removal performances. The bacterial population structure, analyzed by fluorescence in situ hybridization (FISH) with oligonucleotides probes complementary to regions of the 16S and 23S rRNAs, was associated with the P removal performance of the reactor. At one stage of the reactor operation, chemical characterization revealed that extremely poor P removal was occurring. However, like in typical P-removing sludges, complete anaerobic uptake of the carbon substrate occurred. Bacteria inhibiting P removal overwhelmed the reactor, and according to FISH, bacteria of the beta subclass of the class Proteobacteria other than beta-1 or beta-2 were dominant in the sludge (58% of the population). Changes made to the operation of the reactor led to the development of a biomass population with an extremely good P removal capacity. The biochemical transformations observed in this sludge were characteristic of typical P-removing activated sludge. The microbial population analysis of the P-removing sludge indicated that bacteria of the beta-2 subclass of the class Proteobacteria and actinobacteria were dominant (55 and 35%, respectively), therefore implicating bacteria from these groups in high-performance P removal. The changes in operation that led to the improved performance of the reactor included allowing the pH to rise during the anaerobic period, which promoted anaerobic phosphate release and possibly caused selection against non-phosphate-removing bacteria.     

Phylogeography of post-Pleistocene population expansion in a fungus-gardening ant and its microbial mutualists

Although historical biogeographical forces, such as climate-driven range shifts, greatly influence the present-day population genetic structure of animals and plants, the extent to which they affect microbial communities remains largely unknown. We examined the effect of postglacial expansion on the population structure of the northern fungus-gardening ant Trachymyrmex septentrionalis and compared it with that of its two microbial mutualists: a community of lepiotaceous fungal cultivars and associated antibiotic-producing Pseudonocardia bacteria. The ant population genetic structure showed signs of population expansion and subdivision into eastern and western phylogroups that likely originated in the Pleistocene - a pattern shared by many other North American taxa found in the same region. Although dispersal limitation was present in all three symbionts, as suggested by genetic isolation increasing with distance, the host's east-west subdivision of population genetic structure was absent from the microbial mutualist populations. While neither the cultivar nor the Pseudonocardia genetic structure was correlated with that of the ants, they were significantly correlated with each other. These results show that biogeographical forces may act differently on macro- and microscopic organisms, even in the extreme case where microbial mutualists are vertically transmitted from generation to generation and share the same joint ecological niche. It may be that historical climate change played a larger role in determining the population structure of the ant hosts, whereas present-day environmental forces, such as pathogen pressure, determine the structure of associated microbial populations.     

Microbial monitoring of the influence of the stabilization degree of sludge when applied to soil

The aim of this study was to establish the degree of stabilization of sludge and its influence on the evolution of total aerobic micro-organisms and those of faecal origin when the sludge is applied to soil. Two different sludges were used, from a food industry (D) and from an urban treatment plant, (E). Both sludges were stabilized aerobically in laboratory conditions. The stabilization process was monitored by following the evolution of pathogenic micro-organisms and the content of total solids and volatile matter in suspension, while on the other hand the microbial evolution of the samples of soil plus sludge and control soil (S) were monitored over an incubation period of 80 days. After 20 or 40 days of incubation, in both sludge treatments the populations of pathogenic micro-organisms of faecal origin were below detectable limits, regardless of the degree of stabilization of the sludge. Soil without sludge treatment did not show pathogenic micro-organisms.     

PCR typing of tetracycline resistance determinants (Tet A-E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium

The distribution of tetracycline resistance determinants Tet A-E was studied by PCR in 40 tetracycline-resistant Salmonella enterica serotype Hadar (S. hadar) isolates collected from human patients in 1996 and 1997, as well as in the microbial community originating from activated sludges of hospital and urban wastewater treatment facilities. A fast DNA extraction and purification method from activated sludges was used to provide amplifiable DNA. The method is based on the direct lysis of bacteria improved by bead-beating followed by DNA purification on polyvinylpolypyrrolidone spin columns to remove PCR inhibitors. The purified DNAs from salmonellae and activated sludges were characterized for the presence of tetracycline determinants with specific primer pairs designed on the basis of published sequences. The Tet A determinant was present in all clinical isolates and DNAs extracted from the bacterial community of the selected activated sludges. The Tet C determinant was identified in only one of the 40 clinical isolates and in six of the seven environmental samples. No signal was detected for Tet B, D and E determinants. This study revealed a high and stable prevalence of the Tet A determinant in both salmonellae clinical isolates and the microbial community of activated sludges from hospital and urban wastewater treatment facilities over a 2-year period.     

Enhanced Biodegradation and Fate of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) and Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMX) in Anaerobic Soil Slurry Bioprocess

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.     

Effects of mineral nutrients,sludge application rate,and application frequency on biodegradation of two oily sludges

A continuous flow soil respirometer was used to evaluate the effect of nutrient addition, application rate, and application frequency on biodegradation of 2 complex oily sludges in soil. The most rapid biodegradation of the refinery sludge occurred when nitrogen was added to reduce the carbon to nitrogen (C∶N) ratio to 9∶1. The petrochemical sludge was degraded most rapidly when nitrogen, phosphorus, and potassium were added at a rate of 124∶1, C∶NPK; CO₂evolution from both wastes increased with increasing application rates, but the fraction of applied sludge which degraded decreased with increasing application rates. Small frequent applications resulted in a slight increase in respiration rate per unit applied over a single equivalent application, indicating that repeated applications of smaller amounts of sludge result in a more rapid rate of decomposition. The population of total soil bacteria was greatest when 1% of either sludge was added to the soil, whereas 5 and 10% sludge additions resulted in slightly lower microbial populations.     

Among-Population Variation in Microbial Community Structure in the Floral Nectar of the Bee-Pollinated Forest Herb Pulmonaria officinalis L

Background

Microbial communities in floral nectar have been shown to be characterized by low levels of species diversity, yet little is known about among-plant population variation in microbial community composition.

Methodology/Principal Findings

We investigated the microbial community structure (yeasts and bacteria) in floral nectar of ten fragmented populations of the bee-pollinated forest herb Pulmonaria officinalis. We also explored possible relationships between plant population size and microbial diversity in nectar, and related microbial community composition to the distance separating plant populations. Culturable bacteria and yeasts occurring in the floral nectar of a total of 100 plant individuals were isolated and identified by partially sequencing the 16S rRNA gene and D1/D2 domains of the 26S rRNA gene, respectively. A total of 9 and 11 yeast and 28 and 39 bacterial OTUs was found, taking into account a 3% (OTU_0.03) and 1% sequence dissimilarity cut-off (OTU_0.01). OTU richness at the plant population level (i.e. the number of OTUs per population) was low for yeasts (mean: 1.7, range: 0–4 OTUs_0.01/0.03 per population), whereas on average 6.9 (range: 2–13) OTUs_0.03 and 7.9 (range 2–16) OTUs_0.01 per population were found for bacteria. Both for yeasts and bacteria, OTU richness was not significantly related to plant population size. Similarity in community composition among populations was low (average Jaccard index: 0.14), and did not decline with increasing distance between populations.

Conclusions/Significance

We found low similarity in microbial community structure among populations, suggesting that the assembly of nectar microbiota is to a large extent context-dependent. Although the precise factors that affect variation in microbial community structure in floral nectar require further study, our results indicate that both local and regional processes may contribute to among-population variation in microbial community structure in nectar.     

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.     

Impact of an indigenous microbial enhanced oil recovery field trial on microbial community structure in a high pour-point oil reservoir

Based on preliminary investigation of microbial populations in a high pour-point oil reservoir, an indigenous microbial enhanced oil recovery (MEOR) field trial was carried out. The purpose of the study is to reveal the impact of the indigenous MEOR process on microbial community structure in the oil reservoir using 16Sr DNA clone library technique. The detailed monitoring results showed significant response of microbial communities during the field trial and large discrepancies of stimulated microorganisms in the laboratory and in the natural oil reservoir. More specifically, after nutrients injection, the original dominant populations of Petrobacter and Alishewanella in the production wells almost disappeared. The expected desirable population of Pseudomonas aeruginosa, determined by enrichment experiments in laboratory, was stimulated successfully in two wells of the five monitored wells. Unexpectedly, another potential population of Pseudomonas pseudoalcaligenes which were not detected in the enrichment culture in laboratory was stimulated in the other three monitored production wells. In this study, monitoring of microbial community displayed a comprehensive alteration of microbial populations during the field trial to remedy the deficiency of culture-dependent monitoring methods. The results would help to develop and apply more MEOR processes.     

Polar lakes may act as ecological islands to aquatic protists

A fundamental question in ecology is whether microorganisms follow the same patterns as multicellular organisms when it comes to population structure and levels of genetic diversity. Enormous population sizes, predominately asexual reproduction and presumably high dispersal because of small body size could have profound implications on their genetic diversity and population structure. Here, we have analysed the population genetic structure in a lake‐dwelling microbial eukaryote (dinoflagellate) and tested the hypothesis that there is population genetic differentiation among nearby lake subpopulations. This dinoflagellate occurs in the marine‐derived saline lakes of the Vestfold Hills, Antarctica, which are ice‐covered most of the year. Clonal strains were isolated from four different lakes and were genotyped using amplified fragment length polymorphism (AFLP). Our results show high genetic differentiation among lake populations despite their close geographic proximity (<9 km). Moreover, genotype diversity was high within populations. Gene flow in this system is clearly limited, either because of physical or biological barriers. Our results discard the null hypothesis that there is free gene flow among protist lake populations. Instead, limnetic protist populations may differentiate genetically, and lakes act as ecological islands even on the microbial scale.     

Anaerobic microbial counts of different potential anaerobic inocula

Sludges of various origin were analysed on the basis of microbial counts of anaerobes in order to investigate potential and alternative sources for inoculating anaerobic digestors. Sludges from neutralization and oil and fat separation tanks of a sugar cane factory, and from a stabilisation pond of a purified terephthalic-acid-producing plant were found to be adequate potential inocula. Activated sludges were found to have both hydrogenotrophic and acetoclastic methanogens and obligate hydrogen-producing acetogens (OHPA). In all sludges tested, hydrogenotrophic methanogens were the dominant strict anaerobes. The ratio between most probable number counts of acetoclastic methanogens and OPHA seems to be different between dispersed sludges and granular sludges. Correspondence to: J. P. Guyot     

Hydrocarbon degradation capacity and population dynamics of a microbial consortium obtained using a sequencing batch reactor in the presence of molasses

A native microbial consortium capable of degrading hydrocarbons was employed as an inoculum source in a sequencing batch reactor (SBR) using molasses as a carbon source. The microbial biomass in the SBR was able to grow in the presence of molasses, degrading 88% of the reducing sugar. Moreover, the consortium produced in the SBR was capable of maintaining 75% of the capacity for biodegradation of oil with respect to the original capacity of the native microbial consortium. Monitoring of the microbial population structure was accomplished using PCR-DGGE. The results indicated that the microbial populations grown in molasses were stable during crude oil degradation, as judged by comparison to the population structure of the native microbial consortium. The results obtained demonstrated that molasses could be used as a carbon source to promote the growth of biomass with oildegrading capacity.     

Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge.

We investigated the anaerobic biodegradation of mono- and dichlorophenol isomers by fresh (unacclimated) sludge and by sludge acclimated to either 2-chlorophenol, 3-chlorophenol, or 4-chlorophenol. Biodegradation was evaluated by monitoring substrate disappearance and, in selected cases, production of 14CH4 from labeled substrates. In unacclimated sludge, each of the monochlorophenol isomers was degraded. The relative rates of disappearance were in this order: ortho greater than meta greater than para. For the dichlorophenols in unacclimated sludge, reductive dechlorination of the Cl group ortho to phenolic OH was observed, and the monochlorophenol compounds released were subsequently degraded. 3,4-Dichlorophenol and 3,5-dichlorophenol were persistent. Sludge acclimated to 2-chlorophenol cross-acclimated to 4-chlorophenol but did not utilize 3-chlorophenol. This sludge also degraded 2,4-dichlorophenol. Sludge acclimated to 3-chlorophenol cross-acclimated to 4-chlorophenol but not to 2-chlorophenol. This sludge degraded 3,4- and 3,5-dichlorophenol but not 2,3- or 2,5-dichlorophenol. The specific cross-acclimation patterns observed for monochlorophenol degradation demonstrated the existence of two unique microbial activities that were in turn different from fresh sludge. The sludge acclimated to 4-chlorophenol could degrade all three monochlorophenol isomers and 2,4- and 3,4-dichlorophenol. The active microbial population in this sludge appeared to be a mixture of populations present in the 2-chlorphenol- and 3-chlorophenol-acclimated sludges, both of which could utilize 4-chlorophenol. Experiments with 14C-radiolabeled p-chlorophenol, o-chlorophenol, and 2,4-dichlorophenol demonstrated that these compounds were converted to 14CH4 and 14CO2.     

UASB反应器中影响污泥颗粒化的工程因素

研究了具有不同微生物群系的接种污泥、流动方式和流速对上流式厌氧污泥床(UASB)反应器中活性污泥粒化的影响。颗粒化过程包括：微生物絮凝体的形成、亚核的形成,亚核增长和颗粒成熟四个阶段。微絮凝体的形成取决于酸化菌的作用。流体的动量传递和流体对悬浮物的剪切作用是影响亚核形成的关键性工程因素。为此提出最低流速概念,即形成污泥膨胀床的最低流速。合适的进料速率、污泥负荷、布水均匀性以及碱度控制是UASB反应器工程放大和过程控制的四大要素。     

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF,recycling of hydrolytic enzymes and yeast,and recombinant cellulase-producing Aspergillus niger

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.     

Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes

A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.     

Estimation of dominant microbial population sizes in the anaerobic granular sludge of a full-scale UASB treating streptomycin wastewater by PCR-DGGE

Although the dominant members of microbial communities in wastewater bio-treatment systems were often paid attention due to their possible important roles in treatment performance, their population sizes, especially the unculturable species, were still little known. Then PCR-DGGE was used in an attempt to estimate the dominant microbial population sizes in the anaerobic granular sludge treating streptomycin wastewater, coupled with an inoculated strain (Esherichia coli) with known population sizes as an internal standard. The results indicated that the band intensities of the inoculated strain in DGGE profiles showed good correlation with population sizes. Then it was possible to estimate the dominant microbial population sizes by means of comparing their DGGE band intensities with the inoculated strain. The estimated results demonstrated that the sizes of major dominant microbial populations in the sludge sample were at the level of 10⁷–10⁸ CFU/g. The sizes of secondary dominant microbial populations were at the level of 10⁵–10⁶ CFU/g. The microbial populations with the size level lower than 10³ CFU/g were undetectable by PCR-DGGE. These results provided a potential approach to evaluate dominant microbial population sizes in complex microbial communities.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.