Monitoring Diel Variations of Physiological Status and Bacterial Diversity in an Estuarine Microbial Mat: An Integrated Biomarker Analysis

Wieland A., Kühl M.

Short-term effects of irradiance (0-1560 micromol photons m(-2) s(-1)), temperature (10-25 degrees C), and salinity (40-160) on oxygenic photosynthesis and oxygen consumption in a hypersaline mat (Salin-de-Giraud, France) were investigated with microsensors under controlled laboratory conditions. Dark O(2) consumption rates were mainly regulated by the mass transfer limitations imposed by the diffusive boundary layer. Areal rates of net photosynthesis increased with irradiance and saturated at irradiances >400 micromol photons m(-2) s(-1). At low irradiances, oxygen consumption increased more strongly with temperature than photosynthesis, whereas the opposite was observed at saturating irradiances. Net photosynthesis vs. irradiance curves were almost unaffected by decreasing salinity (100 to 40), whereas increasing salinities (100 to 160) led to a decrease of net photosynthesis at each irradiance. Dark O(2) consumption rates, maximal gross and net photosynthesis at light saturation were relatively constant over a broad salinity range (60-100) and decreased at salinities above the in situ salinity of 100. Within the range of natural variation, temperature was more important than salinity in regulating photosynthesis and oxygen consumption. At higher salinities the inhibitory impact of salinity on these processes and therefore the importance of salinity as a regulating environmental parameter increased, indicating that in more hypersaline systems, salinity has a stronger limiting effect on microbial activity.

Jonkers H.M., Koh I.-., Behrend P., Muyzer G., de Beer D.

The sulfate-reducing bacterium strain SRB D2 isolated from the photic zone of a hypersaline microbial mat, from Lake Chiprana, NE Spain, respired pyruvate, alanine, and α-ketoglutarate but not formate, lactate, malate, succinate, and serine at significant rates under fully oxic conditions. Dehydrogenase enzymes of only the former substrates are likely oxygen-tolerant as all substrates supported anaerobic sulfate reduction. No indications were found, however, that aerobic respiration supported growth. Although strain SRB D2 appeared phylogenetically closely related to the oxygen-tolerant sulfate-reducing bacterium Desulfovibrio oxyclinae, substrate spectra were markedly different. Most-probable-number (MPN) estimates of sulfate-reducing bacteria and aerobic heterotrophic bacteria indicated that the latter were numerically dominant in both the photic and aphotic zones of the mat. Moreover, substrate spectra of representative isolates showed that the aerobic heterotrophic bacteria are metabolically more diverse. These findings indicate that sulfate-reducing bacteria in the fully oxic photic zone of mats have to compete with aerobic heterotrophic bacteria for organic substrates. Porewater analysis revealed that total carbohydrates and low-molecular-weight carbon compounds (LMWC) made up substantial fractions of the total dissolved organic carbon (DOC) pool and that nighttime degradation of the former was concomitant with increased concentration of the latter. Our findings indicate that aerobic respiration by sulfate-reducing bacteria contributes to organic carbon mineralization in the oxic zone of microbial mats as daytime porewater LMWC concentrations are above typical half-saturation constants.

Elisabeth Krumbein W., Gorbushina A.A., Holtkamp-Tacken E.

Life and living systems need several important factors to establish themselves and to have a continued tradition. In this article the nature of the borderline situation for microbial life under heavy salt stress is analyzed and discussed using the example of biofilms and microbial mats of sabkha systems of the Red Sea. Important factors ruling such environments are described, and include the following: (1) Microbial life is better suited for survival in extremely changing and only sporadically water-supplied environments than are larger organisms (including humans). (2) Microbial life shows extremely poikilophilic adaptation patterns to conditions that deviate significantly from conditions normal for life processes on Earth today. (3) Microbial life adapts itself to such extremely changing and only ephemerally supportive conditions by the capacity of extreme changes (a) in morphology (pleomorphy), (b) in metabolic patterns (poikilotrophy), (c) in survival strategies (poikilophily), and (d) by trapping and enclosing all necessary sources of energy matter in an inwardly oriented diffusive cycle. All this is achieved without any serious attempt at escaping from the extreme and extremely changing conditions. Furthermore, these salt swamp systems are geophysiological generators of energy and material reservoirs recycled over a geological time scale. Neither energy nor material is wasted for propagation by spore formation. This capacity is summarized as poikilophilic and poikilotroph behavior of biofilm or microbial mat communities in salt and irradiationstressed environmental conditions of the sabkha or salt desert type. We use mainly cyanobacteria as an example, although other bacteria and even eukaryotic fungi may exhibit the same potential of living and surviving under conditions usually not suitable for life on Earth. It may, however, be postulated that such poikilophilic organisms are the true candidates for life support and survival under conditions never recorded on Planet Earth. Mars and some planets of other suns may be good candidates to search for life under conditions normally not thought to be favorable for the maintenance of life.

Navarrete A., Urmeneta J., Cantu J.M., Vegas E., White D.C., Guerrero R.

Villanueva L., Navarrete A., Urmeneta J., White D.C., Guerrero R.

ABSTRACT A combined lipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis was used to monitor changes in the physiological status, biomass, and microbial composition of a microbial mat. In the morning hours, an increase in the biomass of layers containing a high density of phototrophs and a decrease in the growth rate in the deep layers were observed. The combined approach also revealed differences in major groups of microorganisms, including green nonsulfur, gram-positive, and heterotrophic bacteria.

Geyer R., Peacock A.D., White D.C., Lytle C., Van Berkel G.J.

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.

Lauro F.M., Bertoloni G., Obraztsova A., Kato C., Tebo B.M., Bartlett D.H.

Three Clostridium strains were isolated from deep-sea sediments collected at a depth of 6.3–7.3 km in the Japan Trench. Physiological characterization and 16S rDNA analysis revealed that the three isolates were all closely related to Clostridium bifermentans. The spores of all three isolates were resistant to inactivation at high pressure and low temperature. However, despite the fact that the vegetative cells were halotolerant and eurythermal they did not appear to be adapted for growth or viability under the conditions prevailing in the deep-sea sediments from which they were obtained. The results suggest that the isolates had survived as spores in the deep-sea sediments and that the marine benthos could be a source of clostridia originating in other environments.

Peacock A.D., Chang Y.-., Istok J.D., Krumholz L., Geyer R., Kinsall B., Watson D., Sublette K.L., White D.C.

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Program’s Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2–13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

Baker G.C., Smith J.J., Cowan D.A.

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the "universal" primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective "universal" Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.

Hiraishi A., Iwasaki M., Kawagishi T., Yoshida N., Narihiro T., Kato K.

Brisbarre N., Fardeau M., Cueff V., Cayol J., Barbier G., Cilia V., Ravot G., Thomas P., Garcia J., Ollivier B.

A strictly anaerobic, slightly halophilic and moderately thermophilic, sporulating rod designated strain DVird3T was isolated from deep-sea hydrothermal vent samples collected at a depth of approximately 800 m on the Atlantic Ocean Ridge. Strain DVird3T possessed a few laterally inserted flagella, had a DNA G + C content of 33.1 mol% and grew optimally at pH 6.6 and at 45 degrees C. Growth was observed at temperatures between 20 and 58 degrees C and at pH values between 5.8 and 8.2. The optimum NaCl concentration for growth was 3% sea salt (30 g l(-1)); no growth was observed in the presence of 15 or 60 g sea salt l(-1). Strain DVird3T is heterotrophic and utilizes some sugars and various single amino acids. Acetate was the main fatty acid detected from carbohydrate fermentation, together with H2 and CO2. Gelatin was used as an energy source. It performed the Stickland reaction. Phylogenetically, strain DVird3T branched with members of cluster XI of the order Clostridiales, with Clostridium halophilum as its closest relative (similarity of 94.6%). On the basis of its phenotypic, genotypic and phylogenetic characteristics, strain DVird3T (= DSM 15212T = CIP 107654T) is proposed as the type strain of a novel species of the genus Clostridium, Clostridium caminithermale sp. nov.

Spring S., Merkhoffer B., Weiss N., Kroppenstedt R.M., Hippe H., Stackebrandt E.

Taxonomic studies were performed on four strains (D-1/D-an/IIT, C/C-an/B1T, A-1/C-an/C1T and A-1/C-an/IT) of anaerobic, gram-positive, spore-forming bacteria originally isolated from a mat sample retrieved from a shallow, moated area around Lake Fryxell, an Antarctic freshwater lake. Phylogenetic analyses based on 16S rRNA gene sequence data indicated that these strains are affiliated with cluster I clostridia and form a coherent group with Clostridium estertheticum and Clostridium laramiense. Similarity values among 16S rRNA gene sequences within this assemblage ranged between 96.7 and 99.8%. Despite the close phylogenetic relationship, several distinguishing phenotypic traits were found among the novel strains using a polyphasic approach. All strains were psychrophilic, but the temperature optimum for growth differed markedly, ranging from 4 to 16 degrees C. In addition, substrate utilization patterns, fermentation end products, cellular fatty acid profiles and morphological traits enabled a clear differentiation between the strains. DNA-DNA hybridization experiments revealed that each of the four novel strains represents a distinct species, with DNA-DNA similarity values to related strains in the range 16-62%. In contrast, the type strains of C. estertheticum and C. laramiense shared 79% DNA-DNA similarity, indicating a close relationship at the species level. On the basis of genetic and phenotypic properties, it is proposed to designate four novel species of the genus Clostridium to harbour the newly isolated strains: Clostridium frigoris sp. nov. (type strain D-1/D-an/IIT=DSM 14204T=ATCC BMAA-579T), Clostridium lacusfryxellense sp. nov. (type strain C/C-an/B1T=DSM 14205T=ATCC BAA-580T), Clostridium bowmaniisp. nov. (type strain A-1/C-an/C1T=DSM 14206T=ATCC BAA-581T) and Clostridium psychrophilum sp. nov. (type strain A-1/C-an/IT=DSM 14207T=ATCC BAA-582T). It is also proposed to unite C. laramiense and C. estertheticum under C. estertheticum. The subspecies C. estertheticum subsp. laramiense subsp. nov. is established, represented by strain ATCC 51254T (=DSM 14884T). The type strain of C. estertheticum subsp. estertheticum remains NCIMB 12511T (=DSM 8809T).

Jonkers H., Abed R.

Gr�tzschel S., Beer D.

The effects of oxygen concentration on photosynthesis and respiration in two hypersaline cyanobacterial mats were investigated. Experiments were carried out on mats from Eilat, Israel, with moderate photosynthetic activity, and mats from Mallorca, Spain, with high photosynthetic activity. The oxygen concentration in the overlying water above the mats was increased stepwise from 0% to 100% O2. Subsequent changes in oxygen concentration, gross photosynthetic rates, and pH values inside the mats were measured with microelectrodes. According to published reports on the regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme in the CO2-fixation pathway of phototrophs, we expected photosynthetic activity to decrease with increasing oxygen concentration. Gross photosynthetic and total respiration rates in both mats were highest when the O2 concentration was at 0% in the overlying water. Net oxygen production rates under these conditions were the same as under air saturation (21% O2), while gross photosynthetic and respiration rates were lowest at air saturation. In both mats, gross photosynthetic and respiration rates increased upon gradually increasing the oxygen concentration in the overlying water from 21% to 100%. These results contradict the expectation that photosynthesis decreases with increasing oxygen concentration. Increased photosynthetic rates at oxygen concentrations above 21% were probably caused by enhanced oxidation of organic matter and concomitant CO2 production due to the increased oxygen availability. The cause of the high respiration rates at 0% O2 in the overlying water was presumably the enhanced excretion of photosynthetic products during increased photosynthesis. We conclude that the effect of the O2/CO2 concentration ratio on the activity of Rubisco as demonstrated in vitro on enzyme extracts cannot be extrapolated to the situation in intact microbial mats, because the close coupling of the activity of primary producers and heterotrophic bacteria plays a major role in this ecosystem.

Rütters H., Sass H., Cypionka H., Rullkötter J.

In this study, cellular lipid compositions of two mesophilic sulfate-reducing bacteria were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). In Desulfosarcina variabilis and Desulforhabdus amnigenus, alkylether-containing phospholipids were detected which had previously only been found in significant amounts in deeply branching hyperthermophilic bacteria and archaea. Combining information from HPLC-MS analysis and chemical degradation experiments, ether lipids were identified as 1-alkyl-2-acyl-phosphatidyl ethanolamines, glycerols and cholines. In Desulforhabdus amnigenus, n-penta-, n-hexa- and n-heptadecyl ethers were present (in order of decreasing abundance), whereas Desulfosarcina variabilis solely contained n-hexadecyl ether side chains.

Grey A., Costeira R., Lorenzo E., O’Kane S., McCaul M.V., McCarthy T., Jordan S.F., Allen C.C., Kelleher B.P.

AbstractCoastal wetlands are highly efficient ‘blue carbon’ sinks which contribute to mitigating climate change through the long-term removal of atmospheric CO2 and capture of carbon (C). Microorganisms are integral to C sequestration in blue carbon sediments and face a myriad of natural and anthropogenic pressures yet their adaptive responses are poorly understood. One such response in bacteria is the alteration of biomass lipids, specifically through the accumulation of polyhydroxyalkanoates (PHAs) and alteration of membrane phospholipid fatty acids (PLFA). PHAs are highly reduced bacterial storage polymers that increase bacterial fitness in changing environments. In this study, we investigated the distribution of microbial PHA, PLFA profiles, community structure and response to changes in sediment geochemistry along an elevation gradient from intertidal to vegetated supratidal sediments. We found highest PHA accumulation, monomer diversity and expression of lipid stress indices in elevated and vegetated sediments where C, nitrogen (N), PAH and heavy metals increased, and pH was significantly lower. This was accompanied by a reduction in bacterial diversity and a shift to higher abundances of microbial community members favouring complex C degradation. Results presented here describe a connection between bacterial PHA accumulation, membrane lipid adaptation, microbial community composition and polluted C rich sediments. Graphical Abstract Geochemical, microbiological and polyhydroxyalkanoate (PHA) gradient in a blue carbon zone.

Koller M., Mukherjee A., Obruca S., Zinn M.

Among materials emulating fossil plastics in functionality and processability, polyhydroxyalkanoates (PHA) stand out as the sole group that is completely integrated into nature’s closed loop material cycle. Being biobased, biosynthesized, biodegradable, home and industrial compostable, and biocompatible, PHA biopolymers outperform competing polymeric materials labelled with “bio” attributes claiming sustainability. PHA biopolymers exhibit versatile material characteristics mimicking fossil plastics and are the most auspicious candidates to replace established fossil plastics, resins, and fibers. Roughly 40% of all prokaryotic strains accumulate PHA biopolymers, and more than 150 different hydroxyalkanoate (HA) monomers that make up PHA biopolymers have been described, making PHA the most versatile family of biopolymers known to humankind. Commercially relevant PHA homo- and heteropolyesters include 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, and 4-hydroxybutyrate monomers. However, numerous other PHA heteropolyesters having higher number of carbon atoms in the individual building blocks have been studied and found to have reasonably relevant functionalities. Furthermore, PHA biopolymers can also be used in numerous non-plastic applications such as being the source of optically pure chemicals or biologically active substances. Therefore, we currently stand only at the beginning of PHA discovery and industrialization. While reducing plastic pollution, greenhouse gas emissions and climate change are the current drivers for intensified exploration and commercialization of PHA biopolymers, they have a far greater role to play than just being exploited due to their renewable nature and intrinsic biodegradability which have also been reviewed here. As consumers, brand owners, converters, waste managers, and policy makers conceive and acknowledge the beneficial attributes of PHA biopolymers in this current wave of commercialization, the next wave consisting of PHA biopolymers for durable applications would irreversibly reduce fossil plastics use helping us to make a quantum leap in reducing plastic pollution, greenhouse gas emissions, and climate change-related to fossil plastics use.

Wang Y., Liu Y., Wang J., Luo T., Zhang R., Sun J., Zheng Q., Jiao N.

This study investigated the microbial structure in the surface seawater from five coastal sites around Xiamen Island, China, over four seasons to evaluate seasonal environmental fluctuations impact on them. This subtropical island is characterized by long, hot, humid summers, and short, mild, dry winters. All sites were dominated by Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes; microbial community composition was similar across four seasons. However, larger proportions of Gammaproteobacteria and Bacillus were observed during the summer than during any other season. The high ratio of Bacillus, Bacteroidetes, and Clostridia richness to Alphaproteobacteria richness in the summer, suggested that the sites we tested were heavily affected by waste water to other seasons. Correlation-based network analyses among the bacterial species and environmental variables indicated important connections between physiochemical variables and specific taxonomic groups. Collectively, our results suggested that seasonal shifts and wastewater pollution together shape the structures of the microbial communities around Xiamen Island.

Benaiges-Fernandez R., Urmeneta J.

Microbial mats are prokaryotic communities that provide model systems to analyze microbial diversity and ecophysiological interactions. Sulfate-reducing bacteria (SRB) play a key role in sulfur and nutrient recycling in these ecosystems. In this work, specific primers for 16S rRNA encoding gene, previously described, were used to study the diversity of SRB in microbial mats of the Ebro Delta. We confirm that this method is reliable to identify the diversity of SRB in these ecosystems. However, some mismatches in obtained sequences had been observed in our system and must be taken under consideration. Various genera of SRB in Ebro Delta microbial mats were identified, such as Desulfonema, Desulfatitalea, Desulfosalsimonas, Desulfoccocus, and Desulfovibrio. The diversity observed in our samples is very similar to previously reported in other microbial mats communities.

Berlanga M., Palau M., Guerrero R.

Microbial mats are complex biofilms in which the major element cycles are represented at a millimeter scale. In this study, community variability within microbial mats from the Camargue wetlands (Rhone Delta, southern France) were analyzed over 3 years during two different seasons (spring and autumn) and at different layers of the mat (0–2, 2–4, and 4–6 mm). To assess bacterial diversity in the mats, amplicons of the V1–V2 region of the 16S rRNA gene were sequenced. The community’s functionality was characterized using two approaches: (i) inferred functionality through 16S rRNA amplicons genes according to PICRUSt, and (ii) a shotgun metagenomic analysis. Based on the reads distinguished, microbial communities were dominated by Bacteria (~94%), followed by Archaea (~4%) and Eukarya (~1%). The major phyla of Bacteria were Proteobacteria, Bacteroidetes, Spirochaetes, Actinobacteria, Firmicutes, and Cyanobacteria, which together represented 70–80% of the total population detected. The phylum Euryarchaeota represented ~80% of the Archaea identified. These results showed that the total bacterial diversity from the Camargue microbial mats was not significantly affected by seasonal changes at the studied location; however, there were differences among layers, especially between the 0–2 mm layer and the other two layers. PICRUSt and shotgun metagenomic analyses revealed similar general biological processes in all samples analyzed, by season and depth, indicating that different layers were functionally stable, although some taxa changed during the spring and autumn seasons over the 3 years. Several gene families and pathways were tracked with the oxic-anoxic gradient of the layers. Genes directly involved in photosynthesis (KO, KEGG Orthology) were significantly more abundant in the top layer (0–2 mm) than in the lower layers (2–4 and 4–6 mm). In the anoxic layers, the presence of ferredoxins likely reflected the variation of redox reactions required for anaerobic respiration. Sulfatase genes had the highest relative abundance below 2 mm. Finally, chemotaxis signature genes peaked sharply at the oxic/photic and transitional oxic-anoxic boundary. This functional differentiation reflected the taxonomic diversity of the different layers of the mat.

Sathiyanarayanan G., Saibaba G., Kiran G.S., Yang Y., Selvin J.

Hassard F., Gwyther C.L., Farkas K., Andrews A., Jones V., Cox B., Brett H., Jones D.L., McDonald J.E., Malham S.K.

The long term survival of faecal indicator organisms (FIOs) and human pathogenic microorganisms in sediments is important from a water quality, human health and ecological perspective. Typically, both bacteria and viruses strongly associate with particulate matter present in freshwater, estuarine and marine environments. This association tends to be stronger in finer textured sediments and is strongly influenced by the type and quantity of clay minerals and organic matter present. Binding to particle surfaces promotes the persistence of bacteria in the environment by offering physical and chemical protection from biotic and abiotic stresses. How bacterial and viral viability and pathogenicity is influenced by surface attachment requires further study. Typically, long-term association with surfaces including sediments induces bacteria to enter a viable-but-non-culturable (VBNC) state. Inherent methodological challenges of quantifying VBNC bacteria may lead to the frequent under-reporting of their abundance in sediments. The implications of this in a quantitative risk assessment context remain unclear. Similarly, sediments can harbour significant amounts of enteric viruses, however, the factors regulating their persistence remains poorly understood. Quantification of viruses in sediment remains problematic due to our poor ability to recover intact viral particles from sediment surfaces (typically

Lünsmann V., Kappelmeyer U., Taubert A., Nijenhuis I., von Bergen M., Heipieper H.J., Müller J.A., Jehmlich N.

ABSTRACT Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase ( bssA ) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCE Constructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluene-contaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem.

Elling F.J., Becker K.W., Könneke M., Schröder J.M., Kellermann M.Y., Thomm M., Hinrichs K.

The distribution of respiratory quinone electron carriers among cultivated organisms provides clues on both the taxonomy of their producers and the redox processes these are mediating. Our study of the quinone inventories of 25 archaeal species belonging to the phyla Eury-, Cren- and Thaumarchaeota facilitates their use as chemotaxonomic markers for ecologically important archaeal clades. Saturated and monounsaturated menaquinones with six isoprenoid units forming the alkyl chain may serve as chemotaxonomic markers for Thaumarchaeota. Other diagnostic biomarkers are thiophene-bearing quinones for Sulfolobales and methanophenazines as functional quinone analogues of the Methanosarcinales. The ubiquity of saturated menaquinones in the Archaea in comparison to Bacteria suggests that these compounds may represent an ancestral and diagnostic feature of the Archaea. Overlap between quinone compositions of distinct thermophilic and halophilic archaea and bacteria may indicate lateral gene transfer. The biomarker potential of thaumarchaeal quinones was exemplarily demonstrated on a water column profile of the Black Sea. Both, thaumarchaeal quinones and membrane lipids showed similar distributions with maxima at the chemocline. Quinone distributions indicate that Thaumarchaeota dominate respiratory activity at a narrow interval in the chemocline, while they contribute only 9% to the microbial biomass at this depth, as determined by membrane lipid analysis.

Guerrero R., Berlanga M.

Living organisms constantly interact with their habitats, selectively taking up compounds from their surroundings to meet their particular needs but also excreting metabolic products and thus modifying their environment. The small size, ubiquity, metabolic versatility, flexibility, and genetic plasticity (horizontal transfer) of microbes allow them to tolerate and quickly adapt to unfavorable and/or changing environmental conditions. The consumption of resources and the formation of metabolic products by spatially separated microbial populations constitute the driving forces that lead to chemical gradient formation. Communication and cooperation, both within and among bacterial species, have produced the properties that give these organisms a selective advantage. Observations of a wide range of natural habitats have established that bacteria do not function as individuals; rather, the vast majority of bacteria in natural and pathogenic ecosystems live in biofilms, defined as surface-associated, complex aggregates of bacterial communities that are attached to solid substrates and embedded in a polymer matrix of their own production. The spatial configurations of biofilms reach levels of complexity nearing those of multicellular eukaryotes. Microbial consortia have played important roles throughout the history of life on Earth, from the microbial mats (a type of biofilm) that were probably the first ecosystems in the early Archean, to the complex microbiota of the intestinal tract of different animals.

Pärnänen K., Karkman A., Virta M., Eronen-Rasimus E., Kaartokallio H.

Polyhydroxyalkanoates (PHAs) are macromolecules produced by bacteria as means for storing carbon and energy in intracellular granules. PHAs have physical properties similar to those of plastics and have become of interest to industry as materials for environmentally friendly bioplastic production. There is an ongoing search for new PHA-producing bacterial strains and PHA-synthesizing enzymes tolerating extreme conditions to find ways of producing PHAs at cold temperatures and high solute concentrations. Moreover, the study of PHA producers in the sea-ice biome can aid in understanding the microbial ecology of carbon cycling in ice-associated ecosystems. In this study, PHA producers and PHA synthase genes were examined under the extreme environmental conditions of sea ice and cold seawater to find evidence of PHA production in an environment requiring adaptation to high salinity and cold temperatures. Sea ice and cold estuarine water samples were collected from the northern Baltic Sea and evidence of PHA production was gathered, using microscopy with Nile Blue A staining of PHA-granules and PCR assays detecting PHA-synthesis genes. The PHA granules and PHA synthases were found at all sampling locations, in both sea ice and water, and throughout the sampling period spanning over 10 years. Our study shows, for the first time, that PHA synthesis occurs in Baltic Sea cold-adapted bacteria in their natural environment, which makes the Baltic Sea and its cold environments an interesting choice in the quest for PHA-synthesizing bacteria and synthesis genes.

Liang M.C., Ning Z.G., Li Y.K., Song P., Wu N., Yang P.

Reusing reclaimed water for the ecological water has become one of the effective ways to alleviate the water crisis in the rivers and lakes, while its impact on the river and lake ecosystems has become the focus of public attention. The microorganism is one of the most direct and effective monitoring objects, which is generally present in the form of biofilm. Therefore, three kinds of water with different water quality were chosen, which included reclaimed water treated with anaerobic–anoxic–oxic (A2/O) and cyclic activated sludge system process, and surface water, respectively. Glass plates with different roughness (0.1, 1.0 and 10 μm) were used as attaching matrix. Biofilms were cultured with the help of outdoor simulation environment; and the dynamic variation of the biofilm dry weight as well as inorganic and organic components were analyzed. The results showed that the content for dry weight, iron oxide, extracellular polymeric substances (EPS) and chlorophyll a of biofilms attached on multi-media surface in three kinds of water body increased sequentially during the rapid growth period, while the activity of phosphatase fluctuated. Biofilm dry weight and chlorophyll a variation can be described double-constant rate equation (R 2 is over 0.90 and 0.91, respectively), while the iron oxide and EPS content could make use of the parabolic diffusion equation (R 2 is over 0.88 and 0.87, respectively). For the surface of the medium with different roughness, dry weight of the different biofilms with roughness 1.0 μm has the largest content, up to 1.39 mg cm−2. With regard to iron oxide, the content differences are little with different roughness and the maximum content is up to 0.36 μg cm−2, while no significant difference existed between the different water bodies. The contents of EPS, chlorophyll a and phosphatase activity in reclaimed water are higher than that in the surface water, and both of them have the highest content with the roughness 1.0 μm medium surface. Testing phospholipid fatty acids showed that the dominant bacteria are i15: 0, bacteria under two processes of reclaimed water in biofilms were, respectively, higher by 3.7 and 3.5 % than the surface water. Biofilm EPS and chlorophyll a concentration was positively correlated with pH, total phosphorus, COD and Mg2+ concentration in the reclaimed water, and was negatively correlated with NH4+, while the phosphatase activity showed no correlation with water quality indicators. This research aimed to provide a theoretical basis and technical support for health assessment of reclaimed water as the ecological water ecosystem for urban rivers and lakes.

Kunihiro T., Veuger B., Vasquez-Cardenas D., Pozzato L., Le Guitton M., Moriya K., Kuwae M., Omori K., Boschker H.T., van Oevelen D.

Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.

Takasu H., Kunihiro T., Nakano S.

The relationship between bacterial respiratory quinone (RQ) concentration and biomass was assessed for Lake Biwa bacterial assemblages to evaluate the utility of bacterial RQ concentration as an indicator of bacterial carbon. The biomass estimated from the RQ concentration correlated well with that from cell volume, indicating that RQ concentration is an appropriate indicator of bacterial biomass. The estimated carbon content per unit of RQ (carbon conversion factor) of bacteria was 0.67 mg C nmol RQ−1. Bacterial carbon biomass, which was estimated from the RQ concentration using the conversion factor, ranged between 0.008 and 0.054 mg C L−1 (average 0.025 mg C L−1) at 5 m depth and between 0.010 and 0.024 mg C L−1 (average 0.015 mg C L−1) at 70 m depth. Ubiquinone-8-containing bacteria dominated the epilimnion and hypolimnion. Compared to conventional image analysis, bacterial RQ analysis is a less laborious method of simultaneously determining bacterial biomass and community.

Long R.A., Eveillard D., Franco S.L., Reeves E., Pinckney J.L.