Restriction Fragment Length Polymorphism Profiles Research Articles

Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia.

ABSTRACT Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region‐specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX‐1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South‐East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co‐circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region‐specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.

Model development: establishing pigs with homogenous microbial profile in the hind gut

The considerable animal-to-animal variation in microbial profiles is a challenge in elucidating the role of gut microbiota in host metabolism. The main purpose of this study was, therefore, to develop a pig model with reduced animal-to-animal variation in gut microbial profile. Twelve piglets from four sows were reared conventionally and 12 piglets from four sows were reared artificially in high efficiency particulate air (HEPA) filtered isolators. All isolator-reared piglets were given an artificial colostrum formula containing the combined fecal material from all eight sows. All piglets were killed at 21 d of age and intestinal contents subjected to 16s rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) profiling. Resulting T-RFLP profiles clustered into two distinct groups representing the two treatment groups. Furthermore, Bray–Curtis dissimilarity distance values and Dice similarity indices showed reduced beta diversity in isolator-reared pigs indicating animal-to-animal variation was reduced in isolator-reared compared to conventional piglets. However, surprisingly, increased alpha diversity was observed in isolator-reared piglets compared with conventional piglets. In conclusion, the study demonstrated that rearing of piglets under conditions of controlled environment reduced animal-to-animal variation in the hindgut microbiota while paradoxically increasing within animal microbial diversity. Isolator rearing may be useful as a model to improve detection of treatment effects on gut microbiota.

Identification of a 'Candidatus Phytoplasma hispanicum'-related strain, associated with yellows-type diseases, in smoke-tree sharpshooter (Homalodisca liturata Ball).

The 16SrXIII group from phytoplasma bacteria were identified in salivary glands from Homalodisca liturata, which were collected in El Comitán on the Baja California peninsula in Mexico. We were able to positively identify 15 16S rRNA gene sequences with the corresponding signature sequence of 'CandidatusPhytoplasma' (CAAGAYBATKATGTKTAGCYGGDCT) and in silico restriction fragment length polymorphism (RFLP) profiles (F value estimations) coupled with a phylogenetic analysis to confirm their relatedness to 'CandidatusPhytoplasma hispanicum', which in turn belongs to the 16SrXIII group. A restriction analysis was carried out with AluI and EcoRI to confirm that the five sequences belongs to subgroup D. The rest of the sequences did not exhibit any known RFLP profile related to a subgroup reported in the 16SrXIII group.

The effect of vaginal microbial communities on colonization by Staphylococcus aureus with the gene for toxic shock syndrome toxin 1 (TSST-1): a case-control study.

Menstrual toxic shock syndrome is associated with vaginal colonization by Staphylococcus aureus strains that encode toxic shock syndrome toxin 1 (tst+). Interestingly, a small proportion of women are colonized by S. aureus tst+ but do not have symptoms of toxic shock syndrome. Here we sought to determine if differences in the species composition of vaginal bacterial communities reflect a differential risk of colonization by S. aureus capable of producing toxic shock syndrome toxin 1 (TSST-1). The composition of vaginal communities of women that were or were not colonized with S. aureus tst+ were compared based on terminal restriction fragment length polymorphism (T-RFLP) profiles and sequences of cloned 16S rRNA genes. There were no detectable differences in community composition or species rank abundance between communities of women vaginally colonized with S. aureus tst+ as compared to those that were not. Phylogenetic analysis of cloned 16S rRNA gene sequences showed that the predominant members of communities of women colonized with S. aureus tst+ were indistinguishable from those of other healthy women. The data suggest that the numerically dominant members of vaginal communities do not preclude colonization and proliferation of S. aureus tst+ within indigenous microbial communities of the vagina.

Microorganisms reveal what plants do not: wheat growth and rhizosphere microbial communities after Azospirillum brasilense inoculation and nitrogen fertilization under field conditions

Azospirillum brasilense is one of plant growth promoting bacteria used to improve plant growth and grain yield of cereal crops. The level of inoculation response is defined by complex plant-microorganism interactions, many of them still unknown. Thus, we evaluated both agronomic response and microbial ecology of wheat crop under A. brasilense inoculation and nitrogen fertilization at field conditions in order to improve inoculation efficiency. Treatments were: control, nitrogen fertilization and inoculation with 40M and 42M strains. Functional and structural diversity of rhizosphere bacterial communities were evaluated by community-level physiological and terminal restriction fragment length polymorphism profiles. Besides, aerial biomass, grain yield and counts of microaerophilic diazotrophic rhizobacteria were determined. Plant ontogeny modified the number of culturable microaerophilic diazotrophic rhizobacteria. Although agronomic response did not show differences, plant ontogeny and the agricultural practices modified both physiology and genetic structure of rhizosphere microbial communities. Interestingly, these differences due to the treatments were observed at jointing stage but not at grain-filling stage of wheat. Our results demonstrate how different management decisions can change plant- microorganism relationships. While wheat could not show differences between some agricultural treatments, under the soil surface microbial communities could show them.

Molecular identification and prevalence of Acidovorax avenae subsp. avenae causing red stripe of sugarcane in China

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.

“Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma mali” strains infecting sweet and sour cherry in the Czech Republic

AbstractA survey for phytoplasma diseases was conducted in a sweet and sour cherry germplasm collection and in cherry orchards within the Czech Republic during 2014–2015. Phytoplasmas were detected in 21 symptomatic trees. Multiple infections of cherry trees by diverse phytoplasmas of 16SrI and 16SrX groups and 16SrI‐A, 16SrI‐B, 16SrI‐L, 16SrX‐A subgroups were detected by restriction fragment length polymorphism (RFLP). Nevertheless, phylogenetic analysis placed subgroups 16SrI‐B and 16SrI‐L inseparable together onto one branch of phylogenetic tree. This is the first report of subgroups 16SrI‐A and 16SrI‐L in Prunus spp., and subgroup 16SrX‐A in sour cherry trees. Additionally, novel RFLP profiles for 16SrI‐A and 16SrI‐B‐related phytoplasmas were found in cherry samples. Phytoplasmas with these novel profiles belong, however, to their respective 16SrI‐A or 16SrI‐B phylogenetic clades.

Effects of short-term rotation and Trichoderma application on the soil environment and physiological characteristics of Malus hupehensis Rehd. seedlings under replant conditions

In this study, we explored the effects of short-term rotation and Trichoderma application on the plant biomass, root respiration rate, root protective enzymes of Malus hupehensis Rehd., and soil environment under replant conditions, to provide a basis for the prevention of the apple replant disease (ARD). In 2014, old apple orchard soils were treated by rotating peanut, Allium fistulosum L., fallow, and natural grass, and replanted apple orchard soil was used as the control. The soils of the different treatments were mixed well with Trichoderma at a concentration of 0.5% (w/w), and 6kg of each treated soil was added to a pot (outer diameter, 27cm; inner diameter, 23cm; height, 18cm). Uniform seedlings of Malus hupehensis Rehd. were transplanted to the pots on April 23, 2015. Two seedlings were planted in each pot, and 30 pots were prepared for each treatment. The samples were collected at the end of August, when there were visible differences in seedling growth among the treatments. The results showed that rotating Allium fistulosum L. mixed with Trichoderma could significantly improve the populations of bacteria and actinomycetes in the soil, which increased by 189.4% and 107.1%, respectively. The biomass of M. hupehensis significantly improved, and plant fresh weight, dry weight, height, and diameter increased by 186.3%, 205.9%, 58.8%, and 33.2%, respectively. In addition, the total fine root length, total fine root surface area, total fine root volume, and fine root tip number increased by 147.2%, 225.9%, 298.5%, and 331.3%, respectively. Compared with the control seedlings, the treated seedlings showed higher respiration rate of the root system, Superoxide Dismutase activity and Peroxidase activity by 131.0%, 46.3%, and 110%, respectively. Analysis of the terminal restriction fragment length polymorphism profiles showed that the treatment of short-term crop rotation and Trichoderma changed the soil fungal community structure; improved the Simpson index of the soil fungal community; and reduced the Shannon index, Pielou index, and Margalef index. The results suggest that short-term rotation of Allium fistulosum L. mixed with Trichoderma could alleviate ARD.

Phototrophic bacteria dominate consortia, potentially to remove CO2 and H2S from biogas under microaerophilic conditions

The use of microbial consortia to remove contaminants in industrial systems and in natural environments could be an alternative to the use of unique strains of microorganisms, since microbial consortia have greater robustness to environmental fluctuations. However, it is necessary to evaluate the relationship between the genetic structure and functionality of the consortia. In this work, the functional and structural stability over time of two bacterial consortia (C5 and C6) with the potential to remove CO2 and H2S from biogas was evaluated. Both consortia decreased the dissolved CO2 by over 30% at the end of the incubation period, but C5 presented shorter removal kinetics (3.9 days) than C6 (6.4 days). Additionally, a chemical oxidation of H2S could have occurred in the microcosms. Moreover, both consortia presented a stable genetic structure, measured by terminal restriction fragment length polymorphism profiles of the 16S rRNA gene, characterized by high homogeneity and prevalence of the genus Rhodopseudomonas throughout the incubation period, and an increasing abundance of Xanthobacter during the exponential phase of the growth curve in C5, which would account for the functionality of the consortia.

New subgroup 16SrXI-F phytoplasma strain associated with sugarcane grassy shoot (SCGS) disease in India.

A novel phytoplasma subgroup strain SC-Phy385 (GenBank accession number HF586648) was found associated with Sugarcane Grassy Shoot (SCGS) disease in India. Sugarcane (Saccharum officinarum L.) leaf samples with 'grassy shoot' symptoms were collected from the western region of Maharashtra state. The analysis based on restriction fragment length polymorphism (RFLP) profiles and 16S rRNA gene sequences indicated that most of the phytoplasma strains belong to the 16SrXI-B group. However, SCGS phytoplasma strain SC-Phy385 showed unique RFLP profiles with a similarity coefficient of 0.87 using online interactive phytoplasma classification tool, iPhyClassifier. The actual and virtual RFLP profiles of SC-Phy385 using amplified products of the 16S rRNA partial gene and its multiple sequence alignment with other RYD group phytoplasma sequences revealed that this strain was different from phytoplasma groups described earlier, representing new subgroup 16SrXI-F. The 16S rRNA gene-based phylogenetic analysis of reported RYD and BGWL group phytoplasmas showed that they are a genetically diverse group of phytoplasmas.

Terminal restriction fragment length polymorphism profiling of bacterial flora derived from single human hair shafts can discriminate individuals.

Human hairs are the trace evidence most commonly encountered at many crime scenes. However, they have not been effectively utilized for actual criminal investigations because of the low accuracy of their morphological inspection, low detection rate of short tandem repeat (STR) typing, and the problem of heteroplasmy in mitochondrial DNA analysis. Here, we examined the possibility of individual discrimination by comparing profiles of bacterial flora on hair. We carried out the profiling of terminal restriction fragment length polymorphisms (T-RFLP) of the amplified bacterial 16S ribosomal RNA (rRNA) gene from hair samples. Compared with existing STR typing methods that use hair roots, this method using hair shafts allowed the detection of stable bacterial DNA. We successfully obtained the T-RFLP profile from single hair shafts of all volunteers tested. The profiles were specific to each individual, and multiple profiles obtained from the individual him/herself showed higher similarity than those from different individuals. These individual-specific profiles were stably obtained from samples from most volunteers, when collected again after 6months. Storage of the collected hair samples at -30°C was effective for obtaining reproducible T-RF profiles. When unidentified hair samples collected in the laboratory were compared with a pre-constructed database, 17 of 22 hairs were assigned to a small group of people, including the corresponding individuals. These results show that T-RFLP analysis of bacterial flora on a hair shaft found at a crime scene could provide useful information for narrowing down a suspect.

Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences

Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.

Geographic and environmental sources of variation in bacterial community composition in a large-scale municipal landfill site in China.

Little is known regarding how bacterial communities assemble at landfill, as well as how the environment shapes the composition of bacterial community. In this study, up to 42 refuse samples from a large-scale landfill in China were physicochemically and phylogenetically investigated. 16S ribosomal RNA (rRNA) gene-based Illumina MiSeq sequencing (nine samples) revealed that representatives of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Bacteroidetes were dominant in the refuse samples, which was similar to a previous study on landfill leachate by using 454 pyrosequencing. Although 741 operational taxonomic units (OTUs) were detected across all nine samples, 6 of these OTUs were detected in all of the data sets, suggesting difference between bacterial community structures. Geographical differences between the samples, irrespective of depths, were revealed by a principal component analysis (PCA) based on the terminal restriction fragment length polymorphism (TRFLP) profiles of 42 refuse samples. Redundancy analysis (RDA) suggested that environmental heterogeneity (pH, landfilling ages, and depths) and the abundance of bacteria (represented by 16S rRNA gene copy numbers) were the main drivers shaping the bacterial community structure.

Incubation of innovative methanogenic communities to seed anaerobic digesters.

The methanogenic communities in alternative inocula and their potential to increase CH4 production in mesophilic and psychrophilic dairy manure-based anaerobic digesters were examined. Quantitative-PCR and terminal restriction fragment length polymorphism (T-RFLP) profiles were used to determine archaeal and methanogenic community changes when three inocula (wetland sediment (WS), landfill leachate (LL), and mesophilic digestate (MD)) were incubated at 15, 25, and 35°C for 91 and 196days. After each incubation period, the inocula were used in biochemical methane potential (BMP) tests at the incubation temperatures. There was no significant correlation between inoculum mcrA gene copy numbers and CH4 produced in BMP tests, suggesting that population size was not a distinguishing characteristic for predicting CH4 production. Archaeal composition in LL and WS reactors generally converged with MD reactors after incubation at 25 and 35°C for 196days. These MD reactors had high relative abundance of TRF 302, likely Methanosaetaceae, and low acetic acid (0.62-1.61mM). At 15°C incubation, most reactors were associated with high acetic acid (1.61-133.6mM) and dominated by TRF 199, likely Methanosarcinaceae. The LL reactor incubated at 25°C for 91days had higher relative abundance of TRF 199 and produced significantly higher CH4 than WS and MD reactors in BMP test. In the future, it may be possible to create enrichment cultures that favor particular methanogens and use them as inoculum to benefit digesters at low mesophilic temperatures. Our data provides evidence that tailoring the archaeal community could benefit digesters operating under different conditions.

Effects of seaweed fertilizer on the growth of Malus hupehensis Rehd. seedlings, soil enzyme activities and fungal communities under replant condition

Seaweed and its derivatives are widely used as nutrient supplements, biofertilizers, and biostimulants for soil in agriculture. The aim of this study was to evaluate the effects of seaweed fertilizer on the growth of apple (Malus hupehensis Rehd.) seedlings under replant conditions. We investigated the growth of apple seedlings in replant soil treated with seaweed fertilizer at application rates of 0, 5, 20, and 40 g kg−1. The addition of seaweed fertilizer significantly increased the plant height and dry weight of apple seedlings. Seedlings grown in soil treated with seaweed fertilizer, particularly the dose of 40 g kg−1 soil showed higher activities of antioxidant enzymes including superoxide dismutase, peroxidase and catalase, which was accompanied by lower malondialdehyde accumulation. The activities of soil enzymes (invertase, urease, proteinase and phosphatase) were higher in soil treated with seaweed fertilizer than in control soil. An analysis of terminal restriction fragment length polymorphism profiles showed that the fungal communities differed markedly between the 40 g kg−1 seaweed fertilizer treatments and the 0, 5, and 20 g kg−1 treatments. The highest values of Shannon diversity index, evenness index and richness index were in the 40 g kg−1 seaweed fertilizer treatments, and the lowest values of these indexes were in the control. These results suggested that seaweed fertilizer application at a dose of 40 g kg−1 can improve soil enzymes activities, change the soil fungal communities, and improve soil quality. These changes can promote seedlings growth, increase antioxidant activity, and decrease lipid peroxidation in roots, thereby alleviating apple replant disease.

Archaeal rhizosphere communities differ between the native and invasive lineages of the wetland plant Phragmites australis (common reed) in a Chesapeake Bay subestuary

Phragmites australis is a common wetland plant species worldwide and best known in North America as a persistent invasive species. Only in recent decades was a native lineage, Phragmites australis subsp. americanus, confirmed in North American wetlands. This study investigated whether the two lineages support unique microbial communities in the rhizosphere. Paired stands of the native and invasive lineages were sampled at four locations across a Chesapeake Bay subestuary to assess differences in soil chemical parameters and the microbial community. Microbial characterization included quantification of genes coding for bacterial and archaeal 16S rRNA, archaeal ammonia-monooxygenase, methane co-enzyme A, and particulate methane-monooxygenase. These gene targets helped quantify bacterial biomass, archaeal biomass, ammonia-oxidizers, methanogens, and methane oxidizers, respectively. Bacterial and archaeal community composition was determined by creating terminal restriction fragment length polymorphism profiles of the 16S rRNA, and bacterial and fungal composition was assessed using phospholipid fatty acid profiles. Bacterial biomass and composition did not differ between the two lineages, but significant differences were observed in archaeal community composition. There were significantly more copies of archaeal 16S rRNA genes in invasive (1.8 × 108 g−1 soil) compared to native (7.2 × 107 g−1 soil) lineages (P = 0.04) and composition profiles were significantly different according to PerMANOVA (P = 0.006). Phospholipid fatty acid profiles also differed between invasive and native lineages (P = 0.023). These findings support other studies that have noted the ability of native and invasive Phragmites to differentially affect soil biogeochemistry and suggest that archaea in the rhizosphere of wetland plants may be more sensitive to plant variation than bacteria.

Impact of a Recombinant Biocontrol Bacterium, Pseudomonas fluorescens pc78, on Microbial Community in Tomato Rhizosphere.

Pseudomonas fluorescens pc78 is an effective biocontrol agent for soil-borne fungal diseases. We previously constructed a P43-gfp tagged biocontrol bacteria P. fluorescens pc78-48 to investigate bacterial traits in natural ecosystem and the environmental risk of genetically modified biocontrol bacteria in tomato rhizosphere. Fluctuation of culturable bacteria profile, microbial community structure, and potential horizontal gene transfer was investigated over time after the bacteria treatment to the tomato rhizosphere. Tagged gene transfer to other organisms such as tomato plants and bacteria cultured on various media was examined by polymerase chain reaction, using gene specific primers. Transfer of chromosomally integrated P43-gfp from pc78 to other organisms was not apparent. Population and colony types of culturable bacteria were not significantly affected by the introduction of P. fluorescens pc78 or pc78-48 into tomato rhizosphere. Additionally, terminal restriction fragment length polymorphism profiles were investigated to estimate the influence on the microbial community structure in tomato rhizosphere between non-treated and pc78-48-treated samples. Interestingly, rhizosphere soil treated with strain pc78-48 exhibited a significantly different bacterial community structure compared to that of non-treated rhizosphere soil. Our results suggest that biocontrol bacteria treatment influences microbial community in tomato rhizosphere, while the chromosomally modified biocontrol bacteria may not pose any specific environmental risk in terms of gene transfer.

Methanogen communities in a municipal landfill complex in China.

Landfills are significant global sources of atmospheric methane, but little is known about the ecology and community structure of methanogens in these sites. Here, we investigated the methanogen community based on methyl coenzyme M reductase A gene amplicons in the vertical profiles of three different sites at a municipal landfill complex in China. Links between methanogen communities and refuse properties were explored using multivariate analysis. Clone library results showed that most clones (92%) were related to the hydrogenotrophic methanogens, Methanomicrobiales. Almost all of the Methanomicrobiales clones retrieved in this study are members of the genus Methanoculleus Eight clones were affiliated with the genus Methanofollis The remaining clones were clustered within the genus Methanosarcina Terminal restriction fragment length polymorphism profiles showed that the landfill was predominated by 22 taxa, making up 69%-96% of the community. Of these, a single taxon comprised 36%-65% of the communities across all sites and depths. Principal components analysis separated the methanogen community into three groups, irrespective of site or depth. Redundancy analysis suggested that total phosphorus and pH play roles in structuring methanogen communities in landfills.

Chemical multi-contamination drives benthic prokaryotic diversity in the anthropized Toulon Bay

Investigating the impact of human activities on marine coastal ecosystems remains difficult because of the co-occurrence of numerous natural and human-induced gradients. Our aims were (i) to evaluate the links between the chemical environment as a whole and microbial diversity in the benthic compartment, and (ii) to compare the contributions of anthropogenic and natural chemical gradients to microbial diversity shifts. We studied surface sediments from 54 sampling sites in the semi-enclosed Toulon Bay (NW Mediterranean) exposed to high anthropogenic pressure. Previously published chemical data were completed by new measurements, resulting in an in depth geochemical characterization by 29 representative environmental variables. Bacterial and archaeal diversity was assessed by terminal restriction fragment length polymorphism profiling on a selection of samples distributed along chemical gradients. Multivariate statistical analyses explained from 45% to 80% of the spatial variation in microbial diversity, considering only the chemical variables. A selection of trace metals of anthropogenic origin appeared to be strong structural factors for both bacterial and archaeal communities. Bacterial terminal restriction fragment (T-RF) richness correlated strongly with both anthropogenic and natural chemical gradients, whereas archaeal T-RF richness demonstrated fewer links with chemical variables. No significant decrease in diversity was evidenced in relation to chemical contamination, suggesting a high adaptive potential of benthic microbial communities in Toulon Bay.

Bacterial community composition and fhs profiles of low- and high-ammonia biogas digesters reveal novel syntrophic acetate-oxidising bacteria.

BackgroundSyntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO.ResultsAll methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO.ConclusionsThe phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0454-9) contains supplementary material, which is available to authorized users.