Tannery wastewater treatment coupled with bioenergy production in upflow microbial fuel cell under saline condition.

Tannery industry handling huge amount of leather materials release immense amount of saline organic content wastewater. The present research was focused on the treatment of tannery industrial wastewater in UMFC (upflow microbial fuel cell) under saline condition (4%). The UMFC reactor was operated at different organic load (OL) such as 0.6, 1.2, 1.8 and 2.4 gCOD/L respectively. Total chemical oxygen demand (TCOD) removal at 0.6, 1.2, 1.8 gCOD/L was 87 ± 1.2%, 91 ± 1.2% and 93 ± 1.8% respectively. Soluble chemical oxygen demand (SCOD) removal in UMFC at 0.6, 1.2, 1.8 gCOD/L was 85 ± 0.6%, 88 ± 1.2% and 91 ± 1.8% respectively. Total suspended solids (TSS) removal was 49%, 78%, 81% at 0.6, 1.2, 1.8 gCOD/L OL in UMFC. Further, raise in OL to 2.4 gCOD/L showed decrease in TCOD, SCOD (80% and 72%) and TSS (60%) removal. Maximal power production of 854 mV with corresponding PD (power density) of 462 mW/m2 and CD (current density) of 523 mA/m2 was registered at 1.8 gCOD/L OL in UMFC. Increase in OL to 2.4 gCOD/L revealed decline in energy production to 810 mV with PD (385 mW/m2) and CD (438 mA/m2) in UMFC. Maximal bioaccumulation of chromium (95%) was recorded at 1.8 gCOD/L OL. Among different OL used 1.8 gCOD/L OL was optimum for the treatment of tannery wastewater and energy production. Bacterial community analysis in anode of UMFC revealed the dominance of promising electrogenic halophilic strains such as Ochrobactrum, Marinobacter, Rhodococcus and Bacillus in all the OL. Thus, the research clearly revealed the efficacy of halophilic consortium to treat the saline tannery wastewater coupled with bioenergy production in UMFC.

Bacterial Shifts in Nutrient Solutions Flowing Through Biofilters Used in Tomato Soilless Culture.

In soilless culture, slow filtration is used to eliminate plant pathogenic microorganisms from nutrient solutions. The present study focused on the characterization and the potential functions of microbial communities colonizing the nutrient solutions recycled on slow filters during a whole cultivation season of 7 months in a tomato growing system. Bacterial microflora colonizing the solutions before and after they flew through the columns were studied. Two filters were amended with Pseudomonas putida (P-filter) or Bacillus cereus strains (B-filter), and a third filter was a control (C-filter). Biological activation of filter unit through bacterial amendment enhanced very significantly filter efficacy against plant potential pathogens Pythium spp. and Fusarium oxysporum. However, numerous bacteria (103-104 CFU/mL) were detected in the effluent solutions. The community-level physiological profiling indicated a temporal shift of bacterial microflora, and the metabolism of nutrient solutions originally oriented towards carbohydrates progressively shifted towards degradation of amino acids and carboxylic acids over the 7-month period of experiment. Single-strand conformation polymorphism fingerprinting profiles showed that a shift between bacterial communities colonizing influent and effluent solutions of slow filters occurred. In comparison with influent, 16S rDNA sequencing revealed that phylotype diversity was low in the effluent of P- and C-filters, but no reduction was observed in the effluent of the B-filter. Suppressive potential of solutions filtered on a natural filter (C-filter), where the proportion of Proteobacteria (α- and ß-) increased, whereas the proportion of uncultured candidate phyla rose in P- and B-filters, is discussed.

Seasonal Variation on Microbial Community and Methane Production during Anaerobic Digestion of Cattle Manure in Brazil.

Anaerobic digestion is an alternative method for the treatment of animal manure and wastewater. The anaerobic bioconversion of biomass requires a multi-step biological process, including microorganisms with distinct roles. The diversity and composition of microbial structure in pilot-scale anaerobic digestion operating at ambient temperature in Brazil were studied. Influence of the seasonal and temporal patterns on bacterial and archaeal communities were assessed by studying the variations in density, dynamic and diversity and structure. The average daily biogas produced in the summer and winter months was 18.7 and 16 L day(-1), respectively, and there was no difference in the average methane yield. Quantitative PCR analysis revealed that no differences in abundances and dynamics were found for bacterial communities and the total number of Archaea in different seasons. Analysis of bacterial clone libraries revealed a predominance of Firmicutes (54.5 %/summer and 46.7 %/winter) and Bacteroidetes (31.4 %/summer and 44.4 %/winter). Within the Archaea, the phylum Euryarchaeota was predominant in both digesters. Phylogenetic distribution showed changes in percentage between the phyla identified, but no alterations were recorded in the quality and amount of produced methane or community dynamics. The results may suggest that redundancy of microbial groups may have occurred, pointing to a more complex microbial community in the ecosystem related to this ambient temperature system.

Asymmetrical response of anaerobic digestion microbiota to temperature changes.

In natural settings, anaerobic digestion can take place in a wide temperature range, but industrial digesters are usually operated under either mesophilic (~35 °C) or thermophilic (~55 °C) conditions. The ability of anaerobic digestion microbiota to switch from one operating temperature to the other remains poorly documented. We therefore studied the effect of sudden temperature changes (35 °C/55 °C) in lab-scale bioreactors degrading 13C-labelled cellulose. An asymmetric behaviour was observed. In terms of methane production, after an adaptation period, mesophilic inoculum exhibited a functional resistance to temperature increase but no functional resilience when temperature was reset to 35 °C, while thermophilic inoculum methanogenic activity strongly decreased under mesophilic conditions but partially recovered when temperature was reset to 55 °C. Automated ribosomal intergenic spacer analysis community fingerprints evidenced a strong influence of temperature on microbial diversity, particularly pronounced and persistent for Archaea. Key phylotypes involved in 13C-cellulose degradation were identified with a coupled stable isotope probing (SIP)-16S rDNA pyrotag sequencing approach, suggesting that the hydrolytic and fermentative metabolic functions could be maintained thanks to functional redundancy between members of the class Clostridia, whereas methanogenic activity primarily relied on specialized groups affiliated either to genus Methanosarcina (mesophilic conditions), Methanothermobacter or Methanoculleus (thermophilic conditions) that were irreversibly modified by temperature increase.

Vertebrate bacterial gut diversity: size also matters.

BACKGROUND: One of the central issues in microbial ecology is to understand the parameters that drive diversity. Among these parameters, size has often been considered to be the main driver in many different ecosystems. Surprisingly, the influence of size on gut microbial diversity has not yet been investigated, and so far in studies reported in the literature only the influences of age, diet, phylogeny and digestive tract structures have been considered. This study explicitly challenges the underexplored relationship connecting gut volume and bacterial diversity. RESULTS: The bacterial diversity of 189 faeces produced by 71 vertebrate species covering a body mass range of 5.6 log. The animals comprised mammals, birds and reptiles. The diversity was evaluated based on the Simpson Diversity Index extracted from 16S rDNA gene fingerprinting patterns. Diversity presented an increase along with animal body mass following a power law with a slope z of 0.338 ± 0.027, whatever the age, phylogeny, diet or digestive tract structure. CONCLUSIONS: The results presented here suggest that gut volume cannot be neglected as a major driver of gut microbial diversity. The characteristics of the gut microbiota follow general principles of biogeography that arise in many ecological systems.

Shotgun metaproteomic profiling of biomimetic anaerobic digestion processes treating sewage sludge.

Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).

Only Simpson diversity can be estimated accurately from microbial community fingerprints.

Lalande et al. (Microb. Ecol. 66(3):647-658, 2013) introduced a promising approach to quantify microbial diversity from fingerprinting profiles. Their analysis is based on extrapolating the abundance of the phylotypes detectable in a fingerprint towards the rare phylotypes of the community. By considering a set of reconstructed communities, Lalande et al. obtained a range of estimates for phylotype richness, Shannon diversity and Simpson diversity. They reported narrow ranges indicating accurate estimation, especially for Shannon and Simpson diversities. Here, we show that a much larger set of reconstructed communities than the one considered by Lalande et al. is consistent with the fingerprint. We find that the estimates for phylotype richness and Shannon diversity vary over orders of magnitude, but that the estimates for Simpson diversity are restricted to a narrow range (around 10 %). We conclude that only Simpson diversity can be estimated accurately from fingerprints.

Characterization of the microbial diversity in production waters of mesothermic and geothermic Tunisian oilfields.

The microbial diversity of production waters of five Tunisian oilfields was investigated using Single Strand Conformation Polymorphism (SSCP) technique followed by cloning-sequencing. Dynamics of bacterial populations in production waters collected from four wellheads were also evaluated. For all production water samples collected, DNA from Archaea and Eucarya was not sufficiently abundant to permit detection rRNA genes from these groups by PCR-SSCP. In contrast, the bacterial rRNA genes were detected in all samples, except for samples from DOULEB12 and RAMOURA wells. SSCP profiles attested that two of the studied geothermic wells (ASHTART47 and ASHTART48) had shown a clear change over time, whereas a stable diversity was found with the mesophilic DOULEB well (DL3). PCR amplification of rRNA genes was unsuccessful with samples from DOULEB (DL12) at all three sampling time. The bacterial diversity present in production waters collected from pipelines of SERCINA and LITAYEM oilfields was high, while production waters collected from wellheads (ASHTART and DOULEB) exhibited lower diversity. The partial study of the biodiversity showed a dominance of uncultured bacteria and Pseudomonas genus (class of the Gammaproteobacteria) in three of the studied oilfields (ASHTART47, ASHTART48 and DOULEB3). However, for LITAYEM oilfield, a significant dominance of 5 phyla (Proteobacteria, Thermotogae, Firmicutes, Synergistetes, Bacteroidetes) was shown. Our study gives a real picture of the microbiology of some Tunisian oilfield production waters and shows that some of the sequenced bacterial clones have a great similarity to previous sequenced clones described from other oilfields all over the world, indicating that these ecosystems harbour specific microbial communities. These findings can be considered as an indirect indication of the indigenous origin of these clones.

Biological sludge reduction during abattoir wastewater treatment process using a sequencing batch aerobic system.

Excess sludge disposal during biological treatment of wastewater is subject to numerous constraints, including social, health and regulatory factors. To reduce the amount of excess sludge, coupled processes involving different biological technologies are currently under taken. This work presents a laboratory scale sequencing batch aerobic system included an anaerobic zone for biomass synchronization (SBAAS: sequencing batch aerobic anaerobic system). This system was adopted to reduce sludge production during abattoir wastewater (AW) treatment. The average chemical oxygen demand (COD) removal efficiency of 89% was obtained at a hydraulic retention time (HRT) and a sludge retention time (SRT) of 2 days and 15-20 days, respectively. The comparison of SBAAS performances with a conventional sequencing batch activated sludge system (SBASS) found that the observed biomass production yield (Y(obs)) were in the ranges of 0.26 and 0.7 g suspended solids g(-1) COD removed, respectively. A significant reduction in the excess biomass production of 63% was observed by using the SBAAS. In fact, in the anaerobic zone microorganisms consume the intracellular stocks of energy by endogenous metabolism, which limits biosynthesis and accelerates sludge decay. The single strand conformation polymorphism (SSCP) method was used to study the dynamic and the diversity of bacterial communities. Results showed a significant change in the population structure by including the anaerobic stage in the process, and revealed clearly that the sludge production yield can be correlated with the bacterial communities present in the system.

Diversity of bacterial communities that colonize the filter units used for controlling plant pathogens in soilless cultures.

In recent years, increasing the level of suppressiveness by the addition of antagonistic bacteria in slow filters has become a promising strategy to control plant pathogens in the recycled solutions used in soilless cultures. However, knowledge about the microflora that colonize the filtering columns is still limited. In order to get information on this issue, the present study was carried out over a 4-year period and includes filters inoculated or not with suppressive bacteria at the start of the filtering process (two or three filters were used each year). After 9 months of filtration, polymerase chain reaction (PCR)-single strand conformation polymorphism analyses point out that, for the same year of experiment, the bacterial communities from control filters were relatively similar but that they were significantly different between the bacteria-amended and control filters. To characterize the changes in bacterial communities within the filters, this microflora was studied by quantitative PCR, community-level physiological profiles, and sequencing 16SrRNA clone libraries (filters used in year 1). Quantitative PCR evidenced a denser bacterial colonization of the P-filter (amended with Pseudomonas putida strains) than control and B-filter (amended with Bacillus cereus strains). Functional analysis focused on the cultivable bacterial communities pointed out that bacteria from the control filter metabolized more carbohydrates than those from the amended filters whose trophic behaviors were more targeted towards carboxylic acids and amino acids. The bacterial communities in P- and B-filters both exhibited significantly more phylotype diversity and markedly distinct phylogenetic compositions than those in the C-filter. Although there were far fewer Proteobacteria in B- and P-filters than in the C-filter (22% and 22% rather than 69% of sequences, respectively), the percentages of Firmicutes was much higher (44% and 55% against 9%, respectively). Many Pseudomonas species were also found in the bacterial communities of the control filter. The persistence of the amended suppressive-bacteria in the filters is discussed with regards to the management of suppressive microflora in soilless culture.

Study of microbial communities and environmental parameters of seawater collected from three Tunisian fishing harbors in Kerkennah Islands: Statistical analysis of the temporal and spatial dynamics.

Surface seawater, collected from three fishing harbors during different seasons of the years 2015, 2016 and 2017, were assessed for physico-chemical analyses. Results showed that seawater was mainly polluted by hydrocarbons and some heavy metals. Microbial communities' composition and abundance in the studied harbors were performed using molecular approaches. SSCP analysis indicated the presence of Bacteria, Archaea and Eucarya, with dominance of the bacterial domain. Illumina Miseq analysis revealed that the majority of the sequences were affiliated with Bacteria whereas Archaea were detected at low relative abundance. The bacterial community, dominated by Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes, Actinobacteria and Chloroflexi phyla, are known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbons degradation and heavy metals resistance. The main objectives of this study are to assess, for the first time, the organic/inorganic pollution in surface seawater of Kerkennah Islands harbors, and to explore the potential of next generation marine microbiome monitoring to achieve the planning coastal managing strategies worldwide.

Bacterial Consumption of T4 Phages.

The bacterial consumption of viruses not been reported on as of yet even though bacteria feed on almost anything. Viruses are widely distributed but have no acknowledged active biocontrol. Viral biomass undoubtedly reintegrates trophic cycles; however, the mechanisms of this phase still remain unknown. 13C-labelled T4 phages monitor the increase of the density of the bacterial DNA concomitant with the decrease of plaque forming units. We used 12C T4 phages as a control. T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole the source of carbon, nitrogen, and phosphorus. Bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as in the gut or in water.

Molecular and kinetic characterization of mixed cultures degrading natural and synthetic estrogens.

The biodegradation of estradiol (E2), estrone (E1), and ethinylestradiol (EE2) was investigated using mixed bacterial cultures enriched from activated sludge. Enrichments were carried out on E2 or EE2 in batch conditions with acetonitrile as additional carbon source. Degradation experiments were performed both using hormones as sole carbon source or with an additional source. The hormones were completely degraded by these cultures. Estradiol was rapidly converted to E1 within 24 h. Thereafter, E1 degradation began, displaying a lag phase ranging from 3 to 4 days. Estrone depletion took from 48 h to more than 6 days, depending on the culture conditions. For EE2 degradation, when it was the sole carbon source, the lag phase and the time required for its complete removal (7 and 15 days, respectively) were shorter that in cultures with a supplementary carbon source. The specific degradation rates observed for E2 both with and without an additional carbon source were similar. By contrast, the specific degradation rates for E1 and EE2 were, respectively, seven and 20 times faster when these hormones were supplied as the sole carbon source. The bacterial community structure of each culture was characterized by molecular and cultural methods. The mixed cultures were made up of species belonging to Alcaligenes faecalis, Pusillimonas sp., Denitrobacter sp., and Brevundimonas diminuta or related to uncultured Bacteroidetes. The isolated strain B. diminuta achieved the conversion of E2 to E1.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Towards functional molecular fingerprints.

One of the most important challenges in microbial ecology is to determine the ecological function of dominant microbial populations in their environment. In this paper we propose a generic method coupling fingerprinting and mathematical tools to achieve the functional assigning of bacteria detected in microbial consortia. This approach was tested on a nitrification bioprocess where two functions carried out by two different communities could be clearly distinguished. The mathematical theory of observers of dynamical systems has been used to design a dynamic estimator of the active biomass concentration of each functional community from the available measurements on nitrifying performance. Then, the combination of phylotypes obtained by fingerprinting that best approximated the estimated trajectories of each functional biomass was selected through a random optimization method. By this way, a nitritation or nitratation function was assigned to each phylotype detected in the ecosystem by means of functional molecular fingerprints. The results obtained by this approach were successfully compared with the information obtained from 16S rDNA identification. This original approach can be used on any biosystem involving n successive cascading bioreactions performed by n communities.

Diversity of bacteria and fungi in aerosols during screening in a green waste composting plant.

This article outlines a comprehensive analysis of the microbial diversity of aerosols produced during screening in a green waste composting plant using both culture and molecular techniques. Bacteria, thermophilic actinomycetes and fungi were quantified in the aerosols. The structure of the microbial community was examined using a fingerprint technique and DNA libraries. The results show: (i) the very high diversity of bacteria and fungi in aerosols produced during the composting screening stage, (ii) the low percentage of cultivability for bacteria in aerosols, (iii) the abundance of Thermoactinomyces spp. and Aspergillus spp. in compost aerosols.

Influence of abrasion on biofilm detachment: evidence for stratification of the biofilm.

The objective of this paper was to understand the detachment of multispecies biofilm caused by abrasion. By submitting a biofilm to different abrasion strengths (collision of particles), stratification of biofilm cohesion could be highlighted and related to stratification of biofilm bacterial communities using the PCR-SSCP fingerprint method. The biofilm comprised a thick top layer, weakly cohesive and composed of one dominant species, and a thin basal layer, strongly cohesive and composed of a more diverse population. These observations suggest that microbial composition of biofilms may be an important parameter in understanding biofilm detachment.

Investigation of the acclimatization period: example of the microbial aerobic degradation of volatile organic compounds (VOCs).

The aim of this study is to better evaluate the occurrence of an acclimatization-enrichment period, defined as a selection period of consortia having the capability to biodegrade pollutants. In order to perform this evaluation, two experimental strategies were carried out and the results were studied carefully. Two laboratory-scale reactors were inoculated with activated sludge from an urban treatment plant. During the experiment, these reactors were supplied with a gaseous effluent containing VOCs. For both reactors, the composition is different. Three parameters were monitored to characterize the microflora: bacterial activities, bacterial densities, and the genetic structure of Bacteria and Eukarya domains (Single Strand Conformation Polymorphism fingerprint). The obtained results showed that the resultant biodegradation functions were equivalent. The bacterial community structure differs even if six co-migrated peaks were observed. These data suggest that the microbial communities in both reactors were altered differently in response to the treatment but developed a similar capacity to remove VOCs at the issue of this period. Furthermore, it is suggested that the experimental strategies developed in this work lead to an enrichment in terms of functionality and microbial diversity almost equivalent.

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach.

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility.

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.