16S rRNA Gene Copy Numbers Research Articles

Bacterial community activity and dynamics in the biofilm of an experimental hybrid wetland system treating greywater.

The objectives of this study were to determine the biofilm microbial activity and bacterial community structure and successions in greywater treatment filters and to relate the treatment efficiency to the bacterial community parameters. This 10-month study was performed in a newly established experimental system for domestic greywater treatment that consisted of three parallel vertical flow filters (VFs) followed by a horizontal flow filter (HF). A rapid increase in the bacterial community abundance occurred during the first 85days of filter operations, followed by a short-term decrease and the stabilization of the 16S rRNA gene copy numbers at average levels of 1.2 × 109 and 3.2 × 108 copies/g dw in VFs and HF, respectively, until the end of the experiment. The dominant bacterial phyla and genera differed between the VFs and HF. The temporal variation in the bacterial community structure was primarily related to the species replacement, and it was significantly affected by the influent organic carbon and nitrogen compounds in the VFs and the ammonia and organic carbon in the HF filters. Despite the differences in the community structure and assembly mechanisms, the temporal dynamics of the bacterial community showed high congruence between the filter types. The treatment efficiency was related to the biofilm bacterial community diversity and abundance and the abundance of certain bacterial genera in the VF filters. The results suggest that the dominant pathway of nitrogen removal by greywater treatment VFs occurs via coupled heterotrophic nitrification and denitrification, while the contribution of aerobic denitrification is temporally variable in these filters.

Changes in soil microbial community response to precipitation events in a semi-arid steppe of the Xilin River Basin, China

In the context of climate change, precipitation is predicted to become more intense at the global scale. Such change may alter soil microbial communities and the microbially mediated carbon and nitrogen dynamics. In this study, we experimentally repackaged precipitation patterns during the growing season (from June to September) of 2012 in a semi-arid temperate steppe of the Xilin River Basin in Inner Mongolia of China, based on the 60-year growing season precipitation data. Specifically, a total amount of 240 mm simulated precipitation was assigned to experimental plots by taking the following treatments: (1) P6 (6 extreme precipitation events, near the 1st percentile); (2) P10 (10 extreme precipitation events, near the 5th percentile); (3) P16 (16 moderate precipitation events, near the 50th percentile); and (4) P24 (24 events, 60-year average precipitation, near the 50th percentile). At the end of the growing season, we analyzed soil microbial community structure and biomass, bacterial abundance, fungal abundance and bacterial composition, by using phospholipid fatty acid (PLFA), real-time quantitative polymerase chain reaction (RT-qPCR) and 16S rRNA gene clone library methods. The extreme precipitation events did not change soil microbial community structure (represented by the ratio of PLFA concentration in fungi to PLFA concentration in bacteria, and the ratio of PLFA concentration in gram-positive bacterial biomass to PLFA concentration in gram-negative bacterial biomass). However, the extreme precipitation events significantly increased soil microbial activity (represented by soil microbial biomass nitrogen and soil bacterial 16S rRNA gene copy numbers). Soil fungal community showed no significant response to precipitation events. According to the redundancy analysis, both soil microbial biomass nitrogen and soil ammonium nitrogen (NH4-N) were found to be significant in shaping soil microbial community. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla in soil bacterial composition, and responded differently to the extreme precipitation events. Based on the results, we concluded that the extreme precipitation events altered the overall soil microbial activity, but did not impact how the processes would occur, since soil microbial community structure remained unchanged.

Change in microbial communities, soil enzyme and metabolic activity in a Torreya grandis plantation in response to root rot disease

Soil microorganisms have a profound influence on plant growth, but as well on plant disease. It is still not clear, however, how microbial community structures and functional activities are changing in transitions between healthy and diseased soils. We performed detailed bacterial and fungal community analysis in healthy and root-rot diseased soils of Torreya grandis plantation forest in the subtropical region of China. Soil enzyme activities and microbial metabolic profiling was done to find their interactions with the diseased plants and the microbial functioning. The diseased trees showed lower root biomass, total chlorophyll and N contents in leaf, but higher peroxidase activity. The soils under diseased trees had lower soil organic carbon (SOC) content, but higher pH, moisture and available N content. The microbial diversity was unchanged between the healthy and diseased soils, but the diseased soils had lower 16S rRNA and ITS gene copy numbers. Illumina MiSeq sequencing showed that the fungal rather than the bacterial community composition differed significantly (P < 0.05) and Gibberella and Cryptococcus were associated with the diseased trees. The activities of β-D-cellobiosidase, β-glucosidase and peroxidase and microbial carbohydrate utilization rates were typically enhanced in the diseased soils. Changes in SOC, available N and soil moisture associated to root-rot disease were key factors shaping microbial community composition and activity, and closely linked to plant biomass. Our study showed that prolonged root-rot infestation decreased tree vitality and soil microbial biomass, changed fungal community composition and soil functioning towards a faster organic C decomposition, which potentially may increase soil organic matter turnover.

Microbial diversity and antibiotic resistance in a final effluent-receiving lake

Wastewater treatment plants have been recognised as hotspots for antibiotic resistance genes and antibiotic-resistant bacteria which enter the environment. However, the persistence of these genes and bacteria in receiving ecosystems remains poorly understood. The aim of the study was to evaluate the effect of final effluent release on microbial diversity and the antibiotic resistance gene pool in a final effluent-receiving lake. The numbers of total culturable heterotrophs and unculturable bacteria (represented as the 16S rRNA gene copy number) were significantly reduced during the treatment process. The number of ampicillin-resistant bacteria was higher in the sediment than in water samples, suggesting accumulation of ampicillin-resistant bacteria in freshwater sediments. Using an exogenous method, we captured 56 resistance plasmids which were further characterised. Next-generation sequencing revealed that the microbial phyla represented in the studied metagenomes were typical of corresponding environments. The highest relative abundance of antibiotic resistance genes was observed in the final effluent, suggesting that a considerable number of genes were released from the wastewater treatment plant. However, the lowest relative abundance and lowest diversity of the genes in the lake water, compared to the other studied metagenomes, suggest a negligible effect of treated sewage release on antibiotic resistance within water microbial communities of the lake. Furthermore, uncontrolled sewage dumping into this reservoir in the past as well as lower quality of the water upstream of the lake indicated that the wastewater treatment plant protected the studied ecosystem.

Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems.

Airborne bacteria are significantly affected by meteorological and environmental conditions. However, there is little quantitative data available on the effects of these factors on airborne bacteria in urban ecosystems. In the present study, we analyzed weather-dependent changes in the composition of airborne bacterial communities using high throughput sequencing. Samples were collected before and after a period of constant hot weather at four selected sampling sites (YRBS, ZJGUSJC, TJCR, and BLQG) in Hangzhou. Our results show that the average amount of bacterial 16S rRNA gene copy numbers per m3 of air decreased significantly after constant high temperature. In addition, the number of operational taxonomic units and the Shannon–Wiener diversity indexes of the samples at all four selected sampling sites were significantly decreased after the heat event, showing notable impact on bacterial diversity. We also detected a significant increase in the abundances of spore-forming bacteria. Firmicutes increased from 3.7% to 9.9%, Bacillales increased from 2.6% to 7.6%, and Bacillaceae increased from 1.5% to 5.9%. In addition, we observed an increase in beta-Proteobacteria (18.2% to 50.3%), Rhodocyclaceae (6.9% to 29.9%), and Burkholderiaceae (8.1% to 15.2%). On the other hand, the abundance of alpha-Proteobacteria (39.6% to 9.8%), Caulobacteraceae (17.9% to 0.5%), Sphingomonadaceae (7.2% to 3.3%), and Xanthomonadaceae (3.0% to 0.5%) was significantly lower. Taken together, our data suggest that the composition of airborne bacterial communities varies greatly dependent on heat events, and that such communities include several species that are highly susceptible to high-temperature related stressors such as high air temperature, low relative humidity, and high intensity of solar radiation.

English

It is important to understand the Amazonian Dark Earth (ADE) diversified microbial communities which colonize agricultural soils and interact with plants, allowing a more sustainable way of soil utilization. Genomic prospection of biotechnological interest, such as the phenazine biosynthesis genes, was carried out by the characterization of the bacterial community structure through the analysis of the 16S rRNA gene in ADE rhizospheric and bulk soil sampled in the forest, and in the agriculture managed soil, being subsequently cultivated with caupi bean. Additionally, the phzF gene coding for a key enzyme in the phenazine biosynthesis was detected and quantified. Gene polymophism (Terminal Restriction Fragment Length Polymorphism, T-RFLP) analysis revealed differences in the bacterial community structure between colonized rhizospheric and bulk soil, but there were no differences concerning the 16S rRNA gene copy number. Besides, the phzF gene copy number was higher in the rhizospheric than in the bulk soil, without any difference between forest and agricultural soils. This work confirms that the type of soil and the interaction between plants and microorganisms are key factors that shape the structure and diversity of bacterial communities and represent a biotechnological potential, with the possibility of finding natural compounds for use in biological control. Key words: Amazonian Dark Earth, rhizosphere, quantitative polymerase chain reaction (PCR), terminal restriction fragment length polymorphism (T-RFLP), microbial diversity.

Insights into the Populations of Proteolytic and Amino Acid-Fermenting Bacteria from Microbiota Analysis Using In Vitro Enrichment Cultures.

This study evaluated the effects of nitrogen source composition and monensin on the populations of proteolytic and amino acid-fermenting bacteria using in vitro enrichment culture. The experiment was designed with a 2 × 2 factorial arrangement: two nitrogen sources, casein (Cas) and tryptone (Try), and two levels of monensin, 0 (C) and 5µmol/L (M), resulting in four treatments: Cas-C, Cas-M, Try-C, and Try-M. Ruminal fluid collected from three cannulated Holstein dairy cows was used as the inoculum. Each treatment culture was consecutively transferred six times after 24h of incubation. The results showed that ammonia concentration was lower in Cas than in Try, and it was reduced by monensin addition. In the 6th transfer enrichment cultures, the 16S rRNA gene copy numbers of total bacteria were reduced by monensin but was unaffected by nitrogen sources. Principal component analysis showed that the bacterial communities differed among the treatments. At the genus level, Peptostreptococcus accounted for as much as 41% of the total bacteria in Try-C, but it made up less than 0.02% in the other three treatments. A Pearson correlation analysis showed that the relative abundance of Peptostreptococcus was positively correlated with ammonia concentration. Overall, the results suggest that nitrogen source composition and monensin can affect ruminal ammonia production by modulating the ruminal proteolytic bacterial communities, and some hyper-ammonia-producing bacteria of the genus Peptostreptococcus may be among the main culprits contributing to the high ammonia concentration in the rumen.

Ammonia oxidizers in the sea-surface microlayer of a coastal marine inlet.

Planktonic archaea are thought to play an important role in ammonia oxidation in marine environments. Data on the distribution, abundance, and diversity of ammonia oxidizers in the coastal sea-surface microlayer (SML) are lacking, despite previous reports of high abundance of Thaumarchaeota in the SML of estuaries and freshwater lakes. Here, we failed to detect the presence of ammonia-oxidizing bacteria in any of our samples taken from a semi-enclosed marine inlet in Japan. Therefore, we shifted our focus to examine the archaeal community composition as well as the Thaumarchaeota marine group I (MG-I) and ammonia monooxygenase subunit A (amoA) gene copy numbers and composition in the SML and corresponding underlying water (UW, 20 cm). amoA gene copy numbers obtained by quantitative PCR were consistent with the typical values observed in the surface waters of oceanic and coastal environments where nitrification activity has been detected, but the copy numbers were two- to three-fold less than those reported from the surface layers and UW of high mountain lakes. Both amoA and MG-I 16S rRNA gene copy numbers were significantly negatively correlated with chlorophyll-a and transparent exopolymer particle concentrations in the SML. Communities of archaea and ammonia-oxidizing archaea in SML samples collected during low wind conditions (≤5 m s–1) differed the most from those in UW samples, whereas the communities in SML samples collected during high wind conditions were similar to the UW communities. In the SML, low ratios of amoA to MG-I 16S rRNA genes were observed, implying that most of the SML Thaumarchaeota lacked amoA. To our knowledge, our results provide the first comparison of ammonia-oxidizing communities in the coastal SML with those in the UW.

A continuous flow MFC-CW coupled with a biofilm electrode reactor to simultaneously attenuate sulfamethoxazole and its corresponding resistance genes

A continuous flow microbial fuel cell constructed wetland (MFC-CW) coupled with a biofilm electrode reactor (BER) system was constructed to remove sulfamethoxazole (SMX). The BER unit powered by the stacked MFC-CWs was used as a pretreatment unit, and effluent flowed into the MFC-CW for further degradation. The experimental results indicated that the removal rate of 2 or 4 mg/L SMX in a BER unit was nearly 90%, and the total removal rate in the coupled system was over 99%. As the hydraulic retention time (HRT) was reduced from 16 h to 4 h, the SMX removal rate in the BER decreased from 75% to 48%. However, the total removal rate in the coupled system was still over 97%. The maximum SMX removal rate in the MFC-CW, which accounted for 42%–55% of the total removal, was obtained in the anode layer. In addition, the relative abundances of sul genes detected in the systems were in the order of sulI > sulII > sulIII, and significant positive correlations of sul gene copy numbers versus SMX concentration and 16S rRNA gene copy numbers were observed. Furthermore, significant negative correlations were identified between sul genes, 16S rRNA gene copy numbers, and HRT. The abundances of the sul genes in the effluent of the MFC-CW were lower than the abundances observed in the BER effluent. High-throughput sequencing revealed that the microbial community diversity of the BER was affected by running time, power supply forms and HRT. Bio-electricity from the MFC-CW may reduce microbial community diversity and contribute to reduction of the antibiotic resistance gene (ARG) abundance in the BER. Taken together, the BER-MFC-CW coupled system is a potential tool to treat wastewater containing SMX and attenuate corresponding ARG abundance.

Archaea are prominent members of the prokaryotic communities colonizing common forest mushrooms.

In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenarchaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria.

Effective seeding strategy using flat type poly (vinyl alcohol) cryogel for anammox enrichment

In this study, anammox enrichment reactors were operated using flat type poly (vinyl alcohol) cryogel (cryoPVAG) with precultured anammox bacteria (PAB) and activated sludge (AS) from an anoxic tack of the A2O process to evaluate the effect of different seeding sources on anammox enrichment. In addition, cryoPVAGs with different thicknesses (1, 2, and 3 mm) were used to investigate the effects of the thickness on anammox enrichment. The regression analysis with a modified Gompertz model showed that the start-up period of the anammox enrichment using PAB inoculum was approximately 14 days earlier than that of AS inoculum at a nitrogen loading rate of approximately 1 kg-N m−3 day−1. Substrate diffusion was limited in 3-mm cryoPVAG with respect to trend in nitrogen removal rate. Quantitative PCR analysis indicated that in the initial phase, the 16S rRNA gene copy numbers of anammox microorganism in cryoPVAG were significantly different according to the seeding source, but finally converged to a similar level after anammox enrichment. The anammox reaction was initially promoted by cryoPVAG. Next, anammox biomass detached from cryoPVAG and enriched in the bulk phase to maximize NRR. Illumina MiSeq sequencing revealed that Candidatus Brocadia sinica led to the active anammox reaction, and its relative abundance decreased with increasing gel thickness.

AB100. P074. Novel biomarkers for differential diagnosis of intraductal papillary mucinous neoplasms revealed by profiling microbial composition and translocation markers in liquid biopsies

: Pancreatic ductal adenocarcinoma (PDAC) has a major impact on public health, being the fourth-leading cause of cancer-related death in Western countries with a low overall survival rate and rapid deterioration in quality of life. Intraductal papillary mucinous neoplasms (IPMNs) are neoplastic precursor lesions which are understood to evolve from lesions with low-grade dysplasia to high-grade dysplasia to PDAC. Currently, the accuracy of pre-surgery differential diagnosis of cystic lesions is merely 47–78%. However, only after surgical resection of the pancreas can the type of cystic neoplasms and degree of IPMN dysplasia accurately be determined. The discovery of novel biomarkers that improve timely and accurate diagnosis of IPMN and its grade of dysplasia, and thus assist the determination of health risk, are of great importance for better patient management and to minimize diagnostic errors and unnecessary surgical procedures. We hypothesize that gut microbiota may have a role on pancreatic tumorogenesis and progression, given the strong association of oral pathogens such as P. gingivalis and F. nucleatum with systemic inflammation and intestinal cancers. We aimed to (I) determine whether (oral) bacteria are present in pancreatic cystic lesions, and (II) whether cystic microbiota composition associates with IPMN disease severity. We have established a biobank of pancreatic cyst fluid and peripheral blood from patients with pancreatic cystic lesions (n=90, IPMN and benign neoplasms as control) with post-surgery validated diagnosis. The absolute bacterial 16S rRNA gene copy numbers were determined by TaqMan qPCR and microbial compositional profiling by 16S rRNA gene sequencing is being undertaken. Additionally, we measured the magnitude of microbial translocation (MT) inflammation markers in these samples. We found a statistically significant difference of bacterial 16S gene quantities as well as biomarkers of MT and inflammation between different types of pancreatic cystic lesions, as well as significant correlation with IPMN grade of dysplasia. Our finding that several novel biomarkers from liquid biopsies might be used to differentiate between benign and (pre-)malignant pancreatic cystic lesions is of important clinical relevance in diagnosis and treatment for IPMN-related morbidity and mortality, including PDAC.

Use of an improved high-throughput absolute abundance quantification method to characterize soil bacterial community and dynamics

High-throughput sequencing has dramatically expanded our understanding of bacterial communities based on the information of the species types and their relative abundances. Recently, researchers have also become aware of a deficiency in not considering the absolute abundance in this technique. Combining two or more different methods has typically been used to achieve absolute quantification of microbial communities. However, making a combination of different methods not only is time-consuming but also involves potential uncertainty due to variations in the experimental conditions. To simplify the experimental procedure and improve the high-throughput absolute abundance quantification (HAAQ) of a soil bacterial community, we propose an HAAQ method that uses an internal standard strain (ISS) HAAQ-GFP to simultaneously obtain both the relative and absolute abundances in the soil bacterial community. The results showed that a soil bacterial community and its dynamics can be better characterized by the HAAQ method when the optimal concentrations of ISS HAAQ-GFP (105 to 107cellsg−1) were used, and a 16S rRNA gene copy number adjustment was applied. Based on the HAAQ method, we first found that soil bacterial absolute abundances at the genus level fitted well to the partial log-normal distribution function, and most genera concentrations were in the range of 103.5 to 106.5cellsg−1 in the test soils. Our case studies also indicated that more comprehensive descriptions of soil bacterial communities and their dynamics can be achieved by both the relative and absolute abundances than by the relative abundance alone. The improved HAAQ method can be potentially applied to other microbial ecological studies and to stimulating the development of quantitative bacterial ecology studies.

VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing

BackgroundThe 16S rRNA gene-based amplicon sequencing analysis is widely used to determine the taxonomic composition of microbial communities. Once the taxonomic composition of each community is obtained, evolutionary relationships among taxa are inferred by a phylogenetic tree. Thus, the combined representation of taxonomic composition and phylogenetic relationships among taxa is a powerful method for understanding microbial community structure; however, applying phylogenetic tree-based representation with information on the abundance of thousands or more taxa in each community is a difficult task. For this purpose, we previously developed the tool VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community), which is based on the genome-sequenced microbes’ phylogenetic information. Here, we introduce VITCOMIC2, which incorporates substantive improvements over VITCOMIC that were necessary to address several issues associated with 16S rRNA gene-based analysis of microbial communities.ResultsWe developed VITCOMIC2 to provide (i) sequence identity searches against broad reference taxa including uncultured taxa; (ii) normalization of 16S rRNA gene copy number differences among taxa; (iii) rapid sequence identity searches by applying the graphics processing unit-based sequence identity search tool CLAST; (iv) accurate taxonomic composition inference and nearly full-length 16S rRNA gene sequence reconstructions for metagenomic shotgun sequencing; and (v) an interactive user interface for simultaneous representation of the taxonomic composition of microbial communities and phylogenetic relationships among taxa. We validated the accuracy of processes (ii) and (iv) by using metagenomic shotgun sequencing data from a mock microbial community.ConclusionsThe improvements incorporated into VITCOMIC2 enable users to acquire an intuitive understanding of microbial community composition based on the 16S rRNA gene sequence data obtained from both metagenomic shotgun and amplicon sequencing.

Green tea extract and black tea extract differentially influence cecal levels of short-chain fatty acids in rats.

Increasing evidence indicates that gut microbiota plays a critical role to maintain the host's health. The biological function of microbially produced short‐chain fatty acids (SCFA) becomes the focus of attention. This study aimed to compare the effects of green tea extract (GTE) and black tea extract (BTE) on cecal levels of SCFA in rats. Rats consumed an assigned diet of either a control diet, a GTE diet (10 g/kg), or a BTE diet (10 g/kg), for 3 weeks. The dietary addition of GTE significantly reduced the concentrations of acetate and butyrate in cecal digesta compared to the control, but BTE showed an increased trend for a cecal pool. In the GTE group, a significant amount of undigested starch was excreted in feces, but BTE produced no effect. Interestingly, feces of rats fed the BTE diet contained higher bacterial 16S rRNA gene copy numbers for total eubacteria compared to the control diet. Taken together, treatments of the diets with GTE and BTE brought about a different degree of producing SCFA in rat cecum. BTE might advantageously stimulate more SCFA production than GTE by facilitating bacterial utilization of starch.

Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem

The 16S ribosomal RNA gene is the most widely used marker gene in microbial ecology. Counts of 16S sequence variants, often in PCR amplicons, are used to estimate proportions of bacterial and archaeal taxa in microbial communities. Because different organisms contain different 16S gene copy numbers (GCNs), sequence variant counts are biased towards clades with greater GCNs. Several tools have recently been developed for predicting GCNs using phylogenetic methods and based on sequenced genomes, in order to correct for these biases. However, the accuracy of those predictions has not been independently assessed. Here, we systematically evaluate the predictability of 16S GCNs across bacterial and archaeal clades, based on ∼ 6,800 public sequenced genomes and using several phylogenetic methods. Further, we assess the accuracy of GCNs predicted by three recently published tools (PICRUSt, CopyRighter, and PAPRICA) over a wide range of taxa and for 635 microbial communities from varied environments. We find that regardless of the phylogenetic method tested, 16S GCNs could only be accurately predicted for a limited fraction of taxa, namely taxa with closely to moderately related representatives (≲15% divergence in the 16S rRNA gene). Consistent with this observation, we find that all considered tools exhibit low predictive accuracy when evaluated against completely sequenced genomes, in some cases explaining less than 10% of the variance. Substantial disagreement was also observed between tools (R2<0.5) for the majority of tested microbial communities. The nearest sequenced taxon index (NSTI) of microbial communities, i.e., the average distance to a sequenced genome, was a strong predictor for the agreement between GCN prediction tools on non-animal-associated samples, but only a moderate predictor for animal-associated samples. We recommend against correcting for 16S GCNs in microbiome surveys by default, unless OTUs are sufficiently closely related to sequenced genomes or unless a need for true OTU proportions warrants the additional noise introduced, so that community profiles remain interpretable and comparable between studies.

Photoautotrophic organisms control microbial abundance, diversity, and physiology in different types of biological soil crusts

Biological soil crusts (biocrusts) cover about 12% of the Earth’s land masses, thereby providing ecosystem services and affecting biogeochemical fluxes on a global scale. They comprise photoautotrophic cyanobacteria, algae, lichens and mosses, which grow together with heterotrophic microorganisms, forming a model system to study facilitative interactions and assembly principles in natural communities. Biocrusts can be classified into cyanobacteria-, lichen-, and bryophyte-dominated types, which reflect stages of ecological succession. In this study, we examined whether these categories include a shift in heterotrophic communities and whether this may be linked to altered physiological properties. We analyzed the microbial community composition by means of qPCR and high-throughput amplicon sequencing and utilized flux measurements to investigate their physiological properties. Our results revealed that once 16S and 18S rRNA gene copy numbers increase, fungi become more predominant and alpha diversity increases with progressing succession. Bacterial communities differed significantly between biocrust types with a shift from more generalized to specialized organisms along succession. CO2 gas exchange measurements revealed large respiration rates of late successional crusts being significantly higher than those of initial biocrusts, and different successional stages showed distinct NO and HONO emission patterns. Thus, our study suggests that the photoautotrophic organisms facilitate specific microbial communities, which themselves strongly influence the overall physiological properties of biocrusts and hence local to global nutrient cycles.

Role of hemoglobin in hemoglobin-based remediation of the crude oil-contaminated soil

This study investigated the changes in the indigenous microbial community structure with hemoglobin (Hb) application to determine the role of Hb in Hb-based remediation of crude oil-contaminated soil. The phylogenetic diversity of the bacterial community showed that the Hb addition selected surfactants-producing species, thereby, promoting TPH degradation. The significant increase in the CO2 generation, which can be related to the increase in the bacterial abundance inferred from the 16S rRNA gene copy number, supports the enhanced TPH degradation with Hb application. The similar residual TPH concentrations in the presence of only hydrogen peroxide (H2O2) and both Hb and H2O2 suggested that the role of Hb as a catalyst was not as significant as the role of Hb as a nutrient. Also, in the presence of H2O2, a greater recovery of the microbial community structure was observed with the double Hb injection than the single Hb injection. Overall, this study shows that the Hb-based remediation strategies via microbial metabolism can be successfully applied to remediate the crude-oil contaminated Kuwaiti soil.

Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon.

Comparative analyses of microbial structures and gene copy numbers in the anaerobic digestion of various types of sewage sludge

Anaerobic co-digestion of various sewage sludges is a promising approach for greater recovery of energy, but the process is more complicated than mono-digestion of sewage sludge. The applicability of microbial structure analyses and gene quantification to understand microbial conditions was evaluated. The results show that information from gene analyses is useful in managing anaerobic co-digestion and damaged microbes in addition to conventional parameters like total solids, pH and biogas production. Total bacterial 16S rRNA gene copy numbers are the most useful tools for evaluating unstable anaerobic digestion of sewage sludge, rather than mcrA and total archaeal 16S rRNA gene copy numbers, and high-throughput sequencing. First order decay rates of gene copy numbers during pH failure were higher than typical decay rates of microbes in stable operation. The sequencing analyses, including multidimensional scaling, showed very different microbial structure shifts, but the results were not consistent.