Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers

Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.     

Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacter species

The ostrich (Struthio camelus) is a herbivorous bird and although the hindgut is known as the site for fiber digestion, little is known about the microbial diversity in the ostrich hindgut. Our aim was to analyze the microbial diversity in ostrich ceca using a 16S ribosomal RNA gene (rDNA) clone library approach. A total of 310 clones were sequenced and phylogenetically analyzed and were classified into 110 operational taxonomy units (OTUs) based on a 98% similarity criterion. The similarity of the sequences ranged from 86 to 99% and 95 OTUs had less than 98% similarity to the sequences in the public databases. Coverage and the Shannon–Wiener index (H′) of the library were 83.9% and 4.29, respectively. The sequences were assigned to the following 6 phyla: Firmicutes (50.9% of the total number of sequences), Bacteroidetes (39.4%), Fibrobacteres (6.5%), Euryarchaeota (1.9%), Spirochaetes (1.0%), and Verrucomicrobia (0.3%); approximately 90% of the sequences were affiliated with Firmicutes and Bacteroidetes. The only OTU of Fibrobacteres (OTU 107), had 93 and 90% similarity to Fibrobacter succinogenes and F. intestinalis, respectively, suggesting a new species of Fibrobacter in ostrich ceca. Clostridium coccoides and C. leptum formed major groups within the Firmicutes. There was no OTU with high similarity (≥98%) to the 16S rDNA of cultivated fibrolytic bacteria in our library. Although two OTUs were affiliated with Euryarchaeota, no sequence was affiliated with methanogenic Archaea. This study presents the very complex ostrich cecal microbial community, in which the majority of the bacterial species have not yet been cultivated.     

The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis

Aims:  To identify the bacterial and archaeal composition in a mesophilic biogas digester treating pig manure and to compare the consistency of two 16S rDNA-based methods to investigate the microbial structure.
Methods and results:  Sixty-nine bacterial operational taxonomic units (OTU) and 25 archaeal OTU were identified by sequencing two 16S rDNA clone libraries. Most bacterial OTU were identified as phyla of Firmicutes (47·2% of total clones), Bacteroides (35·4%) and Spirochaetes (13·2%). Methanoculleus bourgensis (29·0%), Methanosarcina barkeri (27·4%) and Methanospirillum hungatei (10·8%) were the dominant methanogens. Only 9% of bacterial and 20% of archaeal OTU matched cultured isolates at a similarity index of ≥97%. About 78% of the dominant bacterial (with abundance >3%) and 83% of archaeal OTU were recovered from the denaturing gradient gel electrophoresis (DGGE) bands of V3 regions in 16S rDNAs.
Conclusions:  In the digester, most bacterial and archaeal species were uncultured; bacteria belonging to Firmicutes , Bacteroides and Spirochaetes seem to take charge of cellulolysis, proteolysis, acidogenesis, sulfur-reducing and homoacetogenesis; the most methanogens were typical hydrogenotrophic or hydrogenotrophic/aceticlastic; DGGE profiles reflected the dominant microbiota.
Significance and Impact of the Study:  This study gave a first insight of the overall microbial structure in a rural biogas digester and also indicated DGGE was useful in displaying its dominant microbiota.     

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.     

Comparison of bacterial populations and chemical composition of dairy wastewater held in circulated and stagnant lagoons

AIMS: This study compared the chemical, physical and bacterial composition of circulated and stagnant dairy wastewaters. METHODS AND RESULTS: Samples taken from circulated and stagnant wastewater lagoons, over a 1-year period, were analysed for 10 chemical (total N, NH3, NO3, NO2, Na, Ca, HCO3, Fe, P and K) and six physical (biological oxygen demand, chemical oxygen demand, dissolved solids, electrical conductivity, pH and sodium absorption ratio) parameters and were found to be similar. The 16S rDNA genes from the samples were amplified, cloned and BLAST analysed. In total, 996 stagnant and 1052 circulated wastewater derived sequences were obtained, comprising 294 and 362 operational taxonomic units (OTUs) from the circulated and stagnant wastewaters respectively. Coverage estimates of the OTUs identified were 72.1% for the stagnant, and 63.6% for the circulated wastewater libraries. The greatest difference between the two wastewaters was a c. sixfold greater number of sequences representative of the family Chromatiaceae in the circulated wastewater derived library and a c. fivefold greater number of sequences representative of the phylum Chloroflexi in the stagnant wastewater derived library. CONCLUSIONS: Circulation of dairy wastewater does not affect any of the chemical or physical parameters tested; however, circulation does alter the bacterial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that circulation of dairy wastewater promotes the growth of bacteria within the family Chromatiaceae and that stagnant systems promote the growth of the phylum Chloroflexi.     

Bacterial Population Dynamics in Dairy Waste during Aerobic and Anaerobic Treatment and Subsequent Storage

The objective of this study was to model a typical dairy waste stream, monitor the chemical and bacterial population dynamics that occur during aerobic or anaerobic treatment and subsequent storage in a simulated lagoon, and compare them to those of waste held without treatment in a simulated lagoon. Both aerobic and anaerobic treatment methods followed by storage effectively reduced the levels of total solids (59 to 68%), biological oxygen demand (85 to 90%), and sulfate (56 to 65%), as well as aerobic (83 to 95%), anaerobic (80 to 90%), and coliform (>99%) bacteria. However, only aerobic treatment reduced the levels of ammonia, and anaerobic treatment was more effective at reducing total sulfur and sulfate. The bacterial population structure of waste before and after treatment was monitored using 16S rRNA gene sequence libraries. Both treatments had unique effects on the bacterial population structure of waste. Aerobic treatment resulted in the greatest change in the type of bacteria present, with the levels of eight out of nine phyla being significantly altered. The most notable differences were the >16-fold increase in the phylum Proteobacteria and the approximately 8-fold decrease in the phylum Firmicutes. Anaerobic treatment resulted in fewer alterations, but significant decreases in the phyla Actinobacteria and Bacteroidetes, and increases in the phyla Planctomycetes, Spirochetes, and TM7 were observed.     

Pollution Impacts on Bacterioplankton Diversity in a Tropical Urban Coastal Lagoon System

Despite a great number of published studies addressing estuarine, freshwater and marine bacterial diversity, few have examined urban coastal lagoons in tropical habitats. There is an increasing interest in monitoring opportunistic pathogens as well as indigenous microbial community members in these water bodies by current molecular and microbiological approaches. In this work, bacterial isolates were obtained through selective plate dilution methods to evaluate antibiotic resistances. In addition, 16S rRNA gene libraries were prepared from environmental waters and mixed cultures grown in BHI medium inoculated with Jacarepaguá lagoon waters. Denaturing gradient gel electrophoresis (DGGE) analyses showed distinct community profiles between environmental communities from each studied site and their cultured counterparts. A total of 497 bacterial sequences were analyzed by MOTHUR, yielding 245 operational taxonomic units (OTUs) grouped at 97% similarity. CCA diagrams showcased how several environmental variables affect the distribution of 18 bacterial orders throughout the three distinct habitats. UniFrac metrics and Venn diagrams revealed that bacterial communities retrieved through each experimental approach were significantly different and that only one OTU, closely related to Vibrio cholerae, was shared between them. Potentially pathogenic bacteria were isolated from most sampled environments, fifty percent of which showed antibiotic resistance.     

Comparative metagenomic analysis of bacterial populations in three full-scale mesophilic anaerobic manure digesters

While the use of anaerobic digestion to generate methane as a source of bioenergy is increasing worldwide, our knowledge of the microbial communities that perform biomethanation is very limited. Using next-generation sequencing, bacterial population profiles were determined in three full-scale mesophilic anaerobic digesters operated on dairy farms in the state of Vermont (USA). To our knowledge, this is the first report of a metagenomic analysis on the bacterial population of anaerobic digesters using dairy manure as their main substrate. A total of 20,366 non-chimeric sequence reads, covering the V1-V2 hypervariable regions of the bacterial 16S rRNA gene, were assigned to 2,176 operational taxonomic units (OTUs) at a genetic distance cutoff value of 5 %. Based on their limited sequence identity to validly characterized species, the majority of OTUs identified in our study likely represented novel bacterial species. Using a naïve Bayesian classifier, 1,624 anaerobic digester OTUs could be assigned to 16 bacterial phyla, while 552 OTUs could not be classified and may belong to novel bacterial taxonomic groups that have yet to be described. Firmicutes, Bacteroidetes, and Chloroflexi were the most highly represented bacteria overall, with Bacteroidetes and Chloroflexi showing the least and the most variation in abundance between digesters, respectively. All digesters shared 132 OTUs, which as a “core” group represented 65.4 to 70.6 % of sequences in individual digesters. Our results show that bacterial populations from microbial communities of anaerobic manure digesters can display high levels of diversity despite sharing a common core substrate.     

Blooms of Single Bacterial Species in a Coastal Lagoon of the Southwestern Atlantic Ocean

We investigated seasonal differences in community structure and activity (leucine incorporation) of the planktonic bacterial assemblage in the freshwater and brackish-water zones of a shallow coastal lagoon of the southwestern Atlantic Ocean. Alphaproteobacteria formed the dominant microbial group in both zones throughout the sampling period. After an intrusion of marine water, members of the SAR11 lineage became abundant in the brackish-water zone. These bacteria were apparently distributed over the lagoon during the following months until they constituted almost 30% of all prokaryotic cells at both sampling sites. At the first sampling date (March 2003) a single alphaproteobacterial species unrelated to SAR11, Sphingomonas echinoides, dominated the microbial assemblages in both zones of the lagoon concomitantly with a bloom of filamentous cyanobacteria. Pronounced maxima of leucine incorporation were observed once in each zone of the lagoon. In the freshwater zone, this highly active microbial assemblage was a mix of the typical bacteria lineages expected in aquatic systems. By contrast, a single bacterial genotype with >99% similarity to the facultative pathogen gammaproteobacterial species Stenotrophomonas maltophilia formed >90% of the bacterial assemblage (>10⁷ cell ml⁻¹) in the brackish-water zone at the time point of highest bacterial leucine incorporation. Moreover, these bacteria were equally dominant, albeit less active, in the freshwater zone. Thus, the pelagic zone of the studied lagoon harbored repeated short-term blooms of single bacterial species. This finding may have consequences for environmental protection.     

Bacterial communities in aerosols and manure samples from two different dairies in central and Sonoma valleys of California

Aerosols have been suspected to transport food pathogens and contaminate fruits and vegetables grown in close proximity to concentrated animal feeding operations, but studies are lacking that substantiate such transport. To monitor the potential transport of bacteria originated from fresh or dry manure through aerosols on a dairy, we identified by 16S rRNA sequencing, bacteria in aerosols collected within 2 to 3 meters from dairy cows at two dairies. Gram-positive Firmicutes were predominant in aerosols from a dairy in Sonoma, California, and surrounded by vineyards, in contrast to sequences of Gram-negative Proteobacteria predominant in aerosols from a dairy in Modesto, California, also surrounded by other dairies. Although Firmicutes represented approximately 50% of the 10 most abundant sequences, aerosols from the Sonoma dairy also contained sequences of Bacteriodetes and Actinobacteria, identified previously with animal feces. While none of the top 10 sequences from fresh or dry manure from Modesto dairy were detected in aerosols, two of the sequences from the phylum Bacteriodetes and one from class Clostridia from fresh manure were detected in aerosols from Sonoma. Interestingly, none of the sequences from dry manure were in the top 10 sequences in aerosols from both dairies. The 10 most abundant sequences in aerosols from the Modesto dairy were all from Proteobacteria and nearly half of them were from genus Massilia, which have been isolated previously from immune-compromised people and aerosols. We conclude that the predominant bacteria in aerosols are diverse among locations and that they do not reflect the predominant species of bacteria present in cow feces and/or in close proximity to cows. These results suggest that the aerosol sequences did not originate from manure. Large volumes of aerosols would be required to determine if bacterial sequences from aerosols could be used to track bacteria in manure to crops grown in proximity.     

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.     

Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration

The microbial community of a conventional anaerobic-aerobic sequencing batch reactor was investigated by cloning and sequencing bacterial 16S rDNA. The 92 16S rDNA sequences analysed ranged across 50 different operational taxonomic units (OTU). The majority of these sequences were not closely related to known species. They belonged to 12 different groups, but essentially to the Cytophagales and the Proteobacteria beta, which represented 38% and 17% of the retrieved sequences respectively. No OTU numerically outnumbered the others. However, similarities were observed with previous reports on molecular characterisation of phosphorus-accumulating ecosystems, suggesting an enrichment in microorganisms belonging to the Rhodocyclus group. Thereafter, the ability of this anaerobic-aerobic microbial community to accumulate phosphorus with nitrate as its energy source was investigated. The reactor was shifted from anaerobic-aerobic running conditions to anaerobic-anoxic conditions by injection of nitrate; and its microbial community was monitored by PCR-single strand conformation polymorphism (SSCP). The reactor maintained a good phosphorus accumulation and similar SSCP microbial community patterns for a period of 17 days, suggesting that the same microbial community was able to respire both oxygen and nitrate. However, this situation was unstable, since a breakdown in phosphorus accumulation occurred thereafter.     

Evaluating the prevalence and genetic diversity of adenovirus and polyomavirus in bovine waste for microbial source tracking

This study evaluated and compared the occurrence, concentrations, and genetic diversity of bovine polyomavirus (BPyV) and bovine adenovirus (BAdV) in manure and feces samples for the purpose of determining which of these two viruses is more suitable for bovine fecal indication and microbial source tracking. The comparability and correlation between concentrations/prevalence of these viruses and bacterial fecal indicators (cow-associated Bacteroidetes, Escherichia coli and enterococci) in manure and fecal samples was also determined. A total of 26 dairy manure and 18 individual dairy cow feces samples were tested. The results showed the mean concentration of BAdV in all of dairy manure samples was at least 1 log lower than BPyV (p ≤ 0.005). All of the dairy manure samples tested positive for BPyV but not for BAdV. After combining dairy manure measurements, bacterial indicators had 0.3–0.7 log (p ≤ 0.05) and 1.8–2.2 log (p ≤ 0.005) higher concentrations than BPyV and BAdV, respectively. The concentration of BPyV had a significant positive correlation with the concentration of E. coli and enterococci in the manure samples. The partial VP1 genetic sequences of BPyV isolated from three different farms had a 100% homology to each other and to VP1 sequence (D13942.1) reported in previous study. Based on the occurrence, quantitative and genetic diversity results, BPyV may be a better indicator than BAdV for microbial source tracking at manure application sites.     

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.     

Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.     

Distinct Microbial Communities within the Endosphere and Rhizosphere of Populus deltoides Roots across Contrasting Soil Types

The root-rhizosphere interface of Populus is the nexus of a variety of associations between bacteria, fungi, and the host plant and an ideal model for studying interactions between plants and microorganisms. However, such studies have generally been confined to greenhouse and plantation systems. Here we analyze microbial communities from the root endophytic and rhizospheric habitats of Populus deltoides in mature natural trees from both upland and bottomland sites in central Tennessee. Community profiling utilized 454 pyrosequencing with separate primers targeting the V4 region for bacterial 16S rRNA and the D1/D2 region for fungal 28S rRNA genes. Rhizosphere bacteria were dominated by Acidobacteria (31%) and Alphaproteobacteria (30%), whereas most endophytes were from the Gammaproteobacteria (54%) as well as Alphaproteobacteria (23%). A single Pseudomonas-like operational taxonomic unit (OTU) accounted for 34% of endophytic bacterial sequences. Endophytic bacterial richness was also highly variable and 10-fold lower than in rhizosphere samples originating from the same roots. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Pezizomycotina (40%), while the Agaricomycotina were more abundant in the rhizosphere (34%) than endosphere (17%). Both fungal and bacterial rhizosphere samples were highly clustered compared to the more variable endophyte samples in a UniFrac principal coordinates analysis, regardless of upland or bottomland site origin. Hierarchical clustering of OTU relative abundance patterns also showed that the most abundant bacterial and fungal OTUs tended to be dominant in either the endophyte or rhizosphere samples but not both. Together, these findings demonstrate that root endophytic communities are distinct assemblages rather than opportunistic subsets of the rhizosphere.     

Assessing the impacts of temperature and storage on <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Salmonella</Emphasis>, and <Emphasis Type="Italic">L. monocytogenes</Emphasis> decay in dairy manure

Elevated levels of animal waste-borne pathogen in ambient water is a serious human health issue. Mitigating influx of pathogens from animal waste such as dairy manure to soil and water requires improving our existing knowledge of pathogen reductions in dairy manure treatment methods. This study was conducted to enhance the  understanding of human pathogen decay in liquid dairy manure in anaerobic (AN) and limited aerobic (LA) storage conditions. The decay of three pathogens (Escherichia coli, Salmonella spp., and Listeria monocytogenes) was assessed in bench-scale batch reactors fed with liquid slurry. A series of temperatures (30, 35, 42, and 50 °C) conditions were tested to determine the impacts of temperature on Escherichia coli, Salmonella, and L. monocytogenes decay in AN and LA conditions. Results showed prolonged survival of E. coli compared to Salmonella and L. monocytogenes in both LA and AN environments. Variations in survival among pathogens with temperature and environmental conditions (i.e., LA and AN) indicated the necessity of developing improved dairy manure waste treatment methods for controlling animal waste-borne pathogens. The results of this study will help in improving the current understanding of human pathogen decay in dairy manure for making informed decisions of animal manure treatment by stakeholders.     

Molecular phylogenetic diversity of bacteria and its spatial distribution in composts

AIMS: To investigate microbial diversity of swine manure composts in the initial stage and the spatial distribution due to gradient effect. METHODS AND RESULTS: Samples in different locations of a composting pile were taken and analysed by using a culture-independent approach. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified, cloned, restriction fragment length polymorphism-screened and sequenced. Thirty-three unique sequence types were found among the 110 analysed positive clones from superstratum sample; 56 among 122 from middle-level sample and 32 among 114 from substrate sample, respectively. The sequences related to Clostridium sp. were most common in the composts. One hundred and thirteen out of 121 16S rDNA sequence types displayed homology with those in the GenBank database. Seven 16S rDNA sequence types were not closely related to any known species. The middle-level sample had the highest microbial diversity, containing unique sequences related to Lactosphaera pasteurii, Firmicutes sp., Aerococcus sp., Megasphaera sp. and Stenotrophomonas sp. CONCLUSIONS: Pile temperature significantly affected microbial community in the initial stage of the composting. Microbial community in different locations is quite different resulting from gradient effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study reveal high bacterial diversity in manure composts, and provide molecular evidence to support gradient effect on microbial diversity in initial stage as well.     

Bacterial population dynamics in dairy waste during aerobic and anaerobic treatment and subsequent storage

The objective of this study was to model a typical dairy waste stream, monitor the chemical and bacterial population dynamics that occur during aerobic or anaerobic treatment and subsequent storage in a simulated lagoon, and compare them to those of waste held without treatment in a simulated lagoon. Both aerobic and anaerobic treatment methods followed by storage effectively reduced the levels of total solids (59 to 68%), biological oxygen demand (85 to 90%), and sulfate (56 to 65%), as well as aerobic (83 to 95%), anaerobic (80 to 90%), and coliform (>99%) bacteria. However, only aerobic treatment reduced the levels of ammonia, and anaerobic treatment was more effective at reducing total sulfur and sulfate. The bacterial population structure of waste before and after treatment was monitored using 16S rRNA gene sequence libraries. Both treatments had unique effects on the bacterial population structure of waste. Aerobic treatment resulted in the greatest change in the type of bacteria present, with the levels of eight out of nine phyla being significantly altered. The most notable differences were the >16-fold increase in the phylum Proteobacteria and the approximately 8-fold decrease in the phylum Firmicutes. Anaerobic treatment resulted in fewer alterations, but significant decreases in the phyla Actinobacteria and Bacteroidetes, and increases in the phyla Planctomycetes, Spirochetes, and TM7 were observed.     

Bacterial and archaeal phylogenetic diversity associated with swine sludge from an anaerobic treatment lagoon

Over the last decades, the demand for pork products has increased significantly, along with concern about suitable waste management. Anaerobic-lagoon fermentation for swine-sludge stabilization is a good strategy, although little is known about the microbial communities in the lagoons. Here, we employed a cloning- and sequencing-based analysis of the 16S rRNA gene to characterize and quantify the prokaryotic community composition in a swine-waste-sludge anaerobic lagoon (SAL). DNA sequence analysis revealed that the SAL library harbored 15 bacterial phyla: Bacteroidetes, Cloroflexi, Proteobacteria, Firmicutes, Deinococcus-Thermus, Synergystetes, Gemmatimonadetes, Chlorobi, Fibrobacteres, Verrucomicrobia and candidates division OP5, OP8, WWE1, KSB1, WS6. The SAL library was generally dominated by carbohydrate-oxidizing bacteria. The archaeal sequences were related to the Crenarchaeota and Euryarchaeota phyla. Crenarchaeota predominated in the library, demonstrating that it is not restricted to high-temperature environments, being also responsible for ammonium oxidation in the anaerobic lagoon. Euryarchaeota sequences were associated with the hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales). Quantitative PCR analysis revealed that the number of bacterial cells was at least three orders of magnitude higher than the number of archaeal cells in the SAL. The identified prokaryotic diversity was ecologically significant, particularly the archaeal community of hydrogenotrophic methanogens, which was responsible for methane production in the anaerobic lagoon. This study provided insight into the archaeal involvement in the overall oxidation of organic matter and the production of methane. Therefore, the treatment of swine waste in the sludge anaerobic lagoon could represent a potential inoculum for the start-up of municipal solid-waste digesters.