Changes in microbial community structure and function during vermicomposting of pig slurry

Most studies investigating the effects of earthworms on microorganisms have focused on the changes before and after vermicomposting rather than those that occur throughout the process. In the present study, we designed continuous feeding reactors in which new layers of pig slurry (1.5 and 3 kg) were added sequentially to form an age gradient inside the reactors in order to evaluate the impact of the earthworm species Eisenia fetida on microbial community structure and function. The activity of earthworms greatly reduced the bacterial and fungal biomass and microbial diversity relative to the control values. However, the pronounced presence of earthworms in the younger layers stimulated microbial activity and as such increased carbon mineralization probably due to the fact that the microorganisms may have been less resource-limited as a result of earthworm activity, as indicated by the ratio of monounsaturated to saturated PLFAs.     

Changes in the microbial community during repeated anaerobic microbial dechlorination of pentachlorophenol

Pentachlorophenol (PCP) has been widely used as a pesticide in paddy fields and has imposed negative ecological effect on agricultural soil systems, which are in typically anaerobic conditions. In this study, we investigated the effect of repeated additions of PCP to paddy soil on the microbial communities under anoxic conditions. Acetate was added as the carbon source to induce and accelerate cycles of the PCP degradation. A maximum degradation rate occurred at the 11th cycle, which completely transformed 32.3 μM (8.6 mg L^?1) PCP in 5 days. Illumina high throughput sequencing of 16S rRNA gene was used to profile the diversity and abundance of microbial communities at each interval and the results showed that the phyla of Bacteroidates, Firmicutes, Proteobacteria, and Euryarchaeota had a dominant presence in the PCP-dechlorinating cultures. Methanosarcina, Syntrophobotulus, Anaeromusa, Zoogloea, Treponema, W22 (family of Cloacamonaceae), and unclassified Cloacamonales were found to be the dominant genera during PCP dechlorination with acetate. The microbial community structure became relatively stable as cycles increased. Treponema, W22, and unclassified Cloacamonales were firstly observed to be associated with PCP dechlorination in the present study. Methanosarcina that have been isolated or identified in PCP dechlorination cultures previously was apparently enriched in the PCP dechlorination cultures. Additionally, the iron-cycling bacteria Syntrophobotulus, Anaeromusa, and Zoogloea were enriched in the PCP dechlorination cultures indicated they were likely to play an important role in PCP dechlorination. These findings increase our understanding for the microbial and geochemical interactions inherent in the transformation of organic contaminants from iron rich soil, and further extend our knowledge of the PCP-transforming microbial communities in anaerobic soil conditions.     

Aerobic treatment of pig slurry: microbial aspects

During the treatment of raw pig slurry by batch aerobic fermentation, the presence of surfaces for bacterial attachment resulted in a 1,000-fold increase in the microbial population. In the absence of such surfaces, a 65% reduction of the chemical oxygen demand (COD) of the manure was obtained after 168 h of aeration, whereas a value of 90% was observed in their presence. In the early stages of the treatment, during which the redox potential passed from a value characteristic of anaerobic conditions (-305 mV) to a stable value typical of aerobic conditions (+125 mV) some 120 h later, an amylolytic population dominated by Gram-negative bacteria (chiefly Acinetobacter calcoaceticus) developed; this was followed by a phase in which a proteolytic population tended to become dominant. Populations of some potentially pathogenic bacterial strains characteristic of the porcine intestinal flora declined noticeably during the treatment.     

Bacterial community dynamics during reduction of odorous compounds in aerated pig manure slurry

Aims:  To study the microbial community responsible for the reduction of the polluting load during aerobic digestion of pig slurry.
Methods and Results:  We analysed bacterial succession by nonculture-based methods and determined the physicochemical parameters and polluting substances during 6 days of aerobic digestion. The bacterial subpopulations evolved by aeration, predominantly Bacillus spp., degraded organic matter and vigorously consumed oxygen, as indicated by low oxidation–reduction potential (ORP). In this phase, the volatile fatty acid (VFA) levels drastically decreased, and VFAs were almost depleted on day 4. Simultaneously, the ammonia concentration decreased to its lowest level on day 4; thereafter, it increased until the end of the process. After the decrease in the total organic carbon content in the supernatant of the decomposed slurry, the ORP increased (∼0 mV), and the microbial community showed an abundance of lineages belonging to the phylum Proteobacteria.
Conclusions:  Bacillus was the predominant member of the bacterial community driving the VFA-removal process. Their predominance was related to the presence of available carbon, including VFAs and changes in ORP.
Significance and Impact of the Study:  Information on the relationships among the involved microbes, polluting materials and physicochemical parameters will aid process design and retrofitting of the process.     

Dynamics of microbial community composition and function during in situ bioremediation of a uranium-contaminated aquifer

A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter(-1)). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.     

Adaption of microbial community during the start-up stage of a thermophilic anaerobic digester treating food waste

A successful start-up enables acceleration of anaerobic digestion (AD) into steady state. The microbial community influences the AD performance during the start-up. To investigate how microbial communities changed during the start-up, microbial dynamics was analyzed via high-throughput sequencing in this study. The results confirmed that the AD was started up within 25 d. Thermophilic methanogens and bacterial members functioning in hydrolysis, acidogenesis, and syntrophic oxidation became predominant during the start-up stage, reflecting a quick adaption of microorganisms to operating conditions. Such predominance also indicated the great contribution of these members to the fast start-up of AD. Redundancy analysis confirmed that the bacterial abundance significantly correlated with AD conditions. The stable ratio of hydrogenotrophic methanogens to aceticlastic methanogens is also important to maintain the stability of the AD process. This work will be helpful to understand the contribution of microbial community to the start-up of AD.     

Impact of anaerobic digestion on organic matter quality in pig slurry

Changes in pig slurry organic matter (OM) during anaerobic digestion (AD) were studied in a reactor to characterize OM evolution through AD. OM maturity and stability were evaluated using different biological and physico-chemical methods. Germination and growth chamber experiments revealed a higher maturity of digested slurry (DS) than raw slurry (RS). Soil incubations showed that DS was more stable than RS with a C-mineralization of 12.0 g CO₂-C 100 g^?1 C_org after 49 days as compared to 17.6 g CO₂-C 100 g^?1 C_org. Biochemical fractionation showed a relative increase in stable compounds such as hemicellulose-like and lignin-like molecules. Fourier-transform infrared spectroscopy showed some changes in the chemical structures of OM with a reduction in the aliphatic chain, lipid and polysaccharide levels. A comparison between the evolution of OM during AD and the first weeks of a composting process showed almost identical changes. Finally a theoretical method called Fictitious Atomic-group Separation was applied to the elemental compositions of RS and DS. DS was less humified than RS and presented the properties of a fulvic acid, indicating that the observed stability in DS was mainly due to the biodegradation of the most labile compounds.     

Sulphur fate and anaerobic biodegradation potential during co-digestion of seaweed biomass (Ulva sp.) with pig slurry

Seaweed (Ulva sp.) stranded on beaches were utilized as co-substrate for anaerobic digestion of pig slurry in three-month co-digestion tests in pilot scale anaerobic digesters in the laboratory. The methanogenic potential of Ulva sp. was low compared to that of other potential co-substrates available for use by farmers: 148 N m3CH4/t of volatile solids or 19 N m3CH4/t of crude product. When used as a co-substrate with pig manure (48%/52% w/w), Ulva sp. seaweed did not notably disrupt the process of digestion; however, after pilot stabilisation, biogas produced contained 3.5% H2S, making it unsuitable for energy recovery without treatment. Sequentially addition of the sulphate reduction inhibitor, potassium molybdate, to a final concentration of 3mM, temporarily reduced H2S emissions, but was unable to sustain this reduction over the three-month period. According to these pilot tests, the use of seaweed stranded on beaches as co-substrate in farm-based biogas plants shows some limitations.     

Start-up of anaerobic filters containing different support materials using pig slurry supernatant

Summary Start-up of four laboratory-scale anaerobic filters, containing clay, coral, mussel shell and plastic pall ring support materials, was achieved at a hydraulic retention time of 6 days and a constant COD loading, ab initio, of 5 kg COD.m^–3.d^–1 using a pig slurry supernatant feed. Start-up was most rapid with the clay filter (c. 20 days) and was slowest with the filter containing the mussel shell support. Irrespective of the time taken for start-up, the performance of all four filters at steady-state was similar, with COD removal efficiencies of 69–73% being attained. Start-up and steady-state performance did not correlate directly with either the unit surface area or the porosity of the support materials utilised.     

Relationship between microbial activity and microbial community structure in six full-scale anaerobic digesters

High activity levels and balanced anaerobic microbial communities are necessary to attain proper anaerobic digestion performance. Therefore, this work was focused on the kinetic performance and the microbial community structure of six full-scale anaerobic digesters and one lab-scale co-digester. Hydrolytic (0.6–3.5 g COD g^?1 VSS d^?1) and methanogenic (0.01–0.84 g COD g^?1 VSS d^?1) activities depended on the type of biomass, whereas no significant differences were observed among the acidogenic activities (1.5–2.2 g COD g^?1 VSS d^?1). In most cases, the higher the hydrolytic and the methanogenic activity, the higher the Bacteroidetes and Archaea percentages, respectively, in the biomasses. Hydrogenotrophic methanogenic activity was always higher than acetoclastic methanogenic activity, and the highest values were achieved in those biomasses with lower percentages of Methanosaeta. In sum, the combination of molecular tools with activity tests seems to be essential for a better characterization of anaerobic biomasses.     

Seasonal effects on the survival characteristics of Salmonella Typhimurium and Salmonella Derby in pig slurry during storage

AIMS: Studies were carried out to investigate the survival of Salmonella Typhimurium and Salmonella Derby in pig slurry during summer and winter seasons. METHODS AND RESULTS: Pig slurry samples collected from a commercial fattening house were inoculated with a broth culture of Salmonella Typhimurium and Salmonella Derby, each at a level of log(10) 5.0 CFU ml(-1) and log(10) 2.0 CFU ml(-1). At the higher inoculum level, S. Typhimurium and S. Derby survived for 34 and 23 days, respectively in the summer, and 58 and 46 days, respectively in the winter. Survival at the lower inoculum level for S. Typhimurium and S. Derby was 19 and 16 days, respectively, in the summer and 24 days for both in the winter. CONCLUSIONS: The survival of S. Typhimurium and S. Derby observed in this study indicates that a 2-month holding period of pig slurry, prior to land spreading, may be adequate if separate storage facilities are provided. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite difficulties correlating laboratory studies with on-farm conditions, pig slurry may not represent a major source of transmission of Salmonella spp. in the farm environment in Ireland.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

PLFA profiles for microbial community monitoring in anaerobic digestion

The use of municipal solid waste as feedstock for biogas production offers an interesting possibility for waste treatment with the beneficial effect of gaining a green energy source. The involved processes are very complex, and many different organisms connected via a dynamic food web are associated with them. These complex interactions within these microbial communities are still not clearly understood. Therefore, a phospholipid fatty acid (PLFA) profile analysis method, well established in aerobic but still not as common in anaerobic systems, was used to throw some light on this matter. In the present investigation, a 750 m3 biogas reactor (Roppen, Austria) was monitored over a half-year period. During this period, four different phases in terms of gas production could be determined: low (I), increasing (II), high (III), and decreasing (IV) gas production. In combination with the PLFA profiles, we were able to identify changes in the microbial community associated with these phases.     

Phylogenetic analysis of a microbial community involved in anaerobic oxidation of ammonium nitrogen

The phylogenetic diversity of a microbial community involved in anaerobic oxidation of ammonium nitrogen in the DEAMOX process was studied. Analysis of clone libraries containing 16S rRNA gene inserts of Bacteria, (including Planctomycetes) and Archaea revealed the presence of nucleotide sequences of the microorganisms involved in the main reactions of the carbon, nitrogen, and sulfur cycles, including nitrifying, denitrifying, and ANAMMOX bacteria. In the bacterial clone library, 16S rRNA gene sequences of representatives of the phyla Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Verrucomicrobia, Lentisphaerae, Spirochaetales, and Planctomycetes, as well as of some new groups, were detected. In the archaeal clone library, nucleotide sequences of methanogens belonging to the orders Methanomicrobiales, Methanobacteriales, and Methanosarcinales were found. It is possible that both ANAMMOX bacteria and bacteria of the genus Nitrosomonas are involved in anaerobic ammonium oxidation in the DEAMOX reactor. Many sequences were similar to those from the clone libraries obtained previously from the ANAMMOX community of marine sediments. It is also probable that the DEAMOX reactions occur in natural ecosystems (in marine and freshwater sediments and the oceanic water column), thereby providing for the coupling of the nitrogen and sulfur cycles.     

Characterization of microbial community structure during continuous anaerobic digestion of straw and cow manure

Responses of bacterial and archaeal communities to the addition of straw during anaerobic digestion of manure at different temperatures (37°C, 44°C and 52°C) were investigated using five laboratory-scale semi-continuous stirred tank reactors. The results revealed that including straw as co-substrate decreased the species richness for bacteria, whereas increasing the operating temperature decreased the species richness for both archaea and bacteria, and also the evenness of the bacteria. Taxonomic classifications of the archaeal community showed that Methanobrevibacter dominated in the manure samples, while Methanosarcina dominated in all digesters regardless of substrate. Increase of the operating temperature to 52°C led to increased relative abundance of Methanoculleus and Methanobacterium. Among the bacteria, the phyla Firmicutes and Bacteroidetes dominated within all samples. Compared with manure itself, digestion of manure resulted in a higher abundance of an uncultured class WWE1 and lower abundance of Bacilli. Adding straw to the digesters increased the level of Bacteroidia, while increasing the operating temperature decreased the level of this class and instead increased the relative abundance of an uncultured genus affiliated to order MBA08 (Clostridia). A considerable fraction of bacterial sequences could not be allocated to genus level, indicating that novel phylotypes are resident in these communities.     

Selected microbial consortium of raw and digested pig slurry and its susceptibility to enterocins

A consortium of bacterial genera from raw and digested pig slurry (pig farm at Figa, Slovakia; input and output samples) was counted from February to October 2000. The total counts of enterococci and staphylococci were well-balanced in input samples, with visible reduction of cells in May (3.22 and/or 4.21 log c.f.u./ml). Among organisms important from a sanitary perspective only a slight reduction after standard slurry treatment was found between input and output samples (2.0 log cycles), with no effect in April and May. However, their counts were high (8.1–9.01 log c.f.u./ml). Yersinia sp. were detected in rather high counts (6.47; 6.39 log c.f.u./ml). But these species, as well as pseudomonads and Aeromonas sp. were very effectively reduced by standard slurry treatment. Enterocins (CCM4231, V24 and EC24) produced by our own isolates of enterococci were used to determine the susceptibility of selected microbial strains from slurry to those enterocins. For quantifying the inhibitory activity of enterocins, the titre (expressed in activity-arbitrary units [AU/ml]), corresponding to the reciprocal of the highest dilution showing a distinct inhibition zone of the indicator, was determined. Under the conditions used, enterocins used were active against the selected microbial consortium by activity from 100 up to 800 AU/ml. Moreover, enterocin V24 reduced the growth of Enterobacter cloacae ECL751 as well as Pseudomonas sp. minimally with differences of 1. 54 and 2.2 log cycles.     

Ethanol, BTEX and microbial community interactions in E-blend contaminated soil slurry

Degradation of benzene, toluene, ethylbenzene, m-, p- and o-xylenes (BTEX) and microbial community shifts in soil slurries contaminated with ethanol–gasoline blends (E-blends), containing 10, 50 or 90% (v/v) ethanol (E10, E50 and E90) were studied in soil slurries previously uncontaminated, contaminated by E-blends or ethanol. BTEX originating from E50 degraded fastest whereas from E10 slowest. Among the individual compounds, ethylbenzene degraded fastest (max 30% d⁻¹), and o-xylene slowest (min 1% d⁻¹) during aerobic conditions in previously not contaminated soils. Previous contamination by E-blends increased BTEX degradation significantly (3–19 times) compared with previously uncontaminated soils, whereas previous contamination with ethanol did not show significant difference in BTEX degradation. At least one type of the E-blends during aerobic conditions had a positive effect on total PLFAs (phospholipid fatty acids) and specific PLFAs, i.e. 10Me18:0, 16:1ω6 and cy17:0, but had a negative effect on cy19:0 and 18:2ω6,9c. The effects on total PLFAs, as well as the individual PLFAs, were particularly strong after repeated contamination. The single most affected PLFA was 16:1ω6, which increased 23 times during E10 treatment in soil slurries previously contaminated by E-blends. Altogether, the various E-blends had significantly different effects on BTEX degradation and also on individual PLFAs under aerobic conditions.