Adaptations in bacterial catabolic enzyme activity and community structure in membrane-coupled bioreactors fed simple synthetic wastewater

Membrane-coupled bioreactors (MBRs) offer substantial benefits compared to conventional reactor designs for biological wastewater treatment. MBR treatment efficiency, however, has not been optimized because the effects of the MBR on process microbiology are poorly understood. In this study, the structure and function of the microbial communities growing in MBRs fed simple synthetic wastewater were investigated. In four starch-fed MBRs, the bacterial community substantially increased its alpha-glucosidase affinity (>1000-fold), while the leucine aminopeptidase and heptanoate esterase affinities increased slightly (<40-fold) or remained relatively constant. Concomitant to these physiological adaptations, shifts in the bacterial community structure in two of the starch-fed MBRs were detected by PCR-DGGE. Four of the bacterial populations detected by PCR-DGGE were isolated and exhibited specific growth rates in batch culture ranging from 0.009 to 0.22 h(-1). Our results suggest that bacterial communities growing under increasingly stringent nutrient limitation adapt their enzyme activities primarily for the nutrients provided, but that there is also a more subtle response not linked to the substrates included in the feed medium. Our research also demonstrates that MBRs can support relatively complex bacterial communities even on simple feed media.     

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.     

Aerobic Biological Treatment of Low-Strength Synthetic Wastewater in Membrane-Coupled Bioreactors: The Structure and Function of Bacterial Enrichment Cultures as the Net Growth Rate Approaches Zero

The goal of the current research was to determine if the stringent nutrient limitation imposed by membrane-coupled bioreactors (MBRs) could be used to force mixed bacterial communities to exhibit a zero net growth rate over an extended time period. Mechanistically, this zero net growth rate could be achieved when the amount of energy available for growth is balanced by the maintenance requirements of the bacterial community. Bench-scale MBRs were fed synthetic feed medium containing gelatin as the major organic substrate. Biomass concentrations initially increased rapidly, but subsequently declined until an asymptote was reached. Leucine aminopeptidase activities concomitantly increased by at least 10-fold, suggesting that bacterial catabolic activity remained high even while growth rates became negligible. In contrast, α-glucosidase and heptanoate esterase activities decreased, indicating that the bacterial community specifically adapted to the carbon source in the feed medium. Bacterial community analysis by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments (PCR-DGGE) suggested that the bacterial community structure completely changed from the beginning to the end of each MBR. Excision and nucleotide sequence analysis of prominent PCR-DGGE bands suggested that many of the dominant populations were similar to novel bacterial strains that were previously uncultivated or recently cultivated during studies specifically targeting these novel populations. This research demonstrates that MBRs have substantial practical applications for biological wastewater treatment; in addition, MBRs are a useful tool to study the ecology of slow-growing bacteria.     

Common bacterial responses in six ecosystems exposed to 10 years of elevated atmospheric carbon dioxide

Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys. Representative bacterial 16S rRNA gene clone sequences were deposited in GenBank with Accession No. JQ366086–JQ387568.     

Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction--single-strand conformation polymorphism analysis

The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a culture-independent approach based on single-strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the digester operating at steady state, the 16S rDNA SSCP patterns of the bacterial community showed eight peaks, whereas the 16S rRNA patterns showed six peaks with a very prominent one corresponding to a Spirochaetes-related bacterium. An acidic shock at pH 6 caused an increase in the 16S rRNA level of two Clostridium-related bacteria. After a 1 week starvation period, the major bacteria present reverted to a basal 16S rRNA level proportional to their 16S rDNA level. Starvation revealed the presence of a previously undetected peak whose corresponding sequence was deeply branched into the low G+C Gram-positive bacteria phylum. Twenty-four hours after a spiked addition to the starved digester community of starch, glucose, lactate or sulphate, an upsurge in several new 16S rRNA-derived peaks was observed. Thus, the perturbation approach combined with 16S rRNA analysis revealed bacteria that had not been detected through 16S rDNA analysis.     

Aerobic biological treatment of synthetic municipal wastewater in membrane-coupled bioreactors

Membrane-coupled bioreactors (MBRs) offer many benefits compared to conventional biological wastewater treatment systems; however, their performance characteristics are poorly understood. Laboratory-scale MBRs were used to study bacterial adaptations in physiology and community structure. MBRs were fed a mixture of starch, gelatin, and polyoxyethylene-sorbitan monooleate to simulate the polysaccharide, protein, and lipid components of municipal wastewater. Physiological adaptations were detected by measuring ectoenzyme activity while structural dynamics were studied by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments. As cell biomass accumulated in the MBRs, pollutant removal efficiency initially improved and then stabilized with respect to effluent concentrations of chemical oxygen demand, protein, and carbohydrate. Comparison of the MBR effluent to filtered reactor fluid indicated that a portion of the observed pollutant removal was due to filtration by the membrane rather than microbial activity. The rates of ectoenzyme-mediated polysaccharide (alpha-glucosidase) and protein (leucine aminopeptidase) hydrolysis became relatively constant once pollutant removal efficiency stabilized. However, the maximum rate of lipid hydrolysis (heptanoate esterase) concomitantly increased more than 10-fold. Similarly, alpha-glucosidase and leucine aminopeptidase ectoenzyme affinities were relatively constant, while the heptanoate esterase affinity increased more than 30-fold. Community analysis revealed that a substantial community shift occurred within the first 7 days of operation. A Flavobacterium-like bacterial population dominated the community (>50% of total band intensity) and continued to do so for the remainder of the experiment.     

Bacterial diversity,community structure and potential growth rates along an estuarine salinity gradient

Very little is known about growth rates of individual bacterial taxa and how they respond to environmental flux. Here, we characterized bacterial community diversity, structure and the relative abundance of 16S rRNA and 16S rRNA genes (rDNA) using pyrosequencing along the salinity gradient in the Delaware Bay. Indices of diversity, evenness, structure and growth rates of the surface bacterial community significantly varied along the transect, reflecting active mixing between the freshwater and marine ends of the estuary. There was no positive correlation between relative abundances of 16S rRNA and rDNA for the entire bacterial community, suggesting that abundance of bacteria does not necessarily reflect potential growth rate or activity. However, for almost half of the individual taxa, 16S rRNA positively correlated with rDNA, suggesting that activity did follow abundance in these cases. The positive relationship between 16S rRNA and rDNA was less in the whole water community than for free-living taxa, indicating that the two communities differed in activity. The 16S rRNA:rDNA ratios of some typically marine taxa reflected differences in light, nutrient concentrations and other environmental factors along the estuarine gradient. The ratios of individual freshwater taxa declined as salinity increased, whereas the 16S rRNA:rDNA ratios of only some typical marine bacteria increased as salinity increased. These data suggest that physical and other bottom-up factors differentially affect growth rates, but not necessarily abundance of individual taxa in this highly variable environment.     

Degradation of the Adriatic medusa <Emphasis Type="Italic">Aurelia</Emphasis> sp. by ambient bacteria

The decomposition of jellyfish after major bloom events results in the release of large amounts of nutrients, which can significantly alter nutrient and oxygen dynamics in the surrounding environment. The response of the ambient bacterial community to decomposing jellyfish biomass was evaluated in two marine ecosystems, the Gulf of Trieste (northern Adriatic Sea) and Big Lake (Mljet Island, southern Adriatic Sea). The major difference between these two ecosystems is that Aurelia sp. medusae occur throughout the year in the oligotrophic Big Lake, whereas in the mesotrophic Gulf of Trieste, they occur only seasonally and often as blooms. Addition of homogenized jellyfish to enclosed bottles containing ambient water from each of these systems triggered considerable changes in the bacterial community dynamics and in the nutrient regime. The high concentrations of protein, dissolved organic phosphorous (DOP), and PO₄ ³⁻ immediately after homogenate addition stimulated increase in bacterial abundance and production rate, coupled with NH₄ ⁺ accumulation in both ecosystems. Our preliminary results of the bacterial community structure, as determined with denaturing gradient gel electrophoresis, indicated differences in the bacterial community response between the two ecosystems. Despite divergence in the bacterial community responses to jellyfish homogenate, increased bacterial biomass and growth rates in both distinctive marine systems indicate potentially significant effects of decaying jellyfish blooms on microbial plankton.     

Use of 16S rDNA community fingerprints to study cricket hindgut microbial communities

A nucleic acid-based method was evaluated in the course of a study of microbial community structure in the cricket hindgut. Genomic DNA was extracted from the hindgut microbial community of Acheta domesticus and used as a template in the polymerase chain reaction (PCR) method, using primers that align to well conserved regions of the 16S rRNA gene. The rDNA-PCR product was used as a community probe to generate restriction fragment length polymorphisms (RFLPs) of hindgut bacterial isolates and gut microbial communities of insects fed different diets. Fingerprints of the bacterial isolates consisted of several bands suggesting multiple rRNA operons. In contrast with soil communities, hindgut community RFLP contained distinguishable band patterns. However, community rDNA fingerprints were complex and varied among insects fed similar diets, suggesting considerable intrinsic variability in the hindgut microbial community structure between crickets regardless of dietary regime. These results suggest that community RFLP methods using broad-specific phylogenetic probes do not have the resolution or specificity required to ascertain the effect of diet on the cricket hindgut microbial community structure.     

Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1‐3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance‐decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay.     

Effects of nutrient availability and Ochromonas sp. predation on size and composition of a simplified aquatic bacterial community

Predation and competition are two main factors that determine the size and composition of aquatic bacterial populations. Using a simplified bacterial community, composed of three strains characterized by different responses to predation, a short-term laboratory experiment was performed to evaluate adaptations and relative success in communities with experimentally controlled levels of predation and nutrient availability. A strain with a short generation time (Pseudomonas putida), one with high plasticity in cell morphology (Flectobacillus sp. GC5), and one that develops microcolonies (Pseudomonas sp. CM10), were selected. The voracious flagellate Ochromonas sp. was chosen as a predator. To describe adaptations against grazing and starvation, abundance, biomass and relative heterogeneity of bacteria were measured. On the whole, the strains in the predation-free cultures exhibited unicellular growth, and P. putida represented the largest group. The presence of Ochromonas strongly reduced bacterial abundance, but not always the total biomass. The activity of grazers changed the morphological composition of the bacterial communities. Under grazing pressure the relative composition of the community depended on the substrate availability. In the presence of predators, P. putida abundance declined in both high and low nutrient treatments, and Pseudomonas CM10 developed colonies. Flectobacillus was only numerically codominant in the nutrient-rich environments.     

Biodegradation of organic wastes containing surfactants in a biomass recycle reactor.

The microbial biodegradation of simulated graywater, containing 21.5 mg of linear alkylbenzene sulfonate liter-1, was investigated with a continuous-flow bioreactor with 100% biomass recycle. Low concentrations of organic matter in the ultrafiltration eluate were achieved by hydraulic residence times as short as 1.6 h and for periods of up to 74 days at a hydraulic residence time of 6 h. Upon a shift from the chemostat to the biomass recycle mode, the increase in biomass with time approximated a linear rather than an exponential function. Biomass densities as high as 6.8 g of cell protein liter-1 were reached; this was 50-fold higher than the steady-state biomass level in chemostats fed the same medium. We assessed physiological changes in the microbial community after a switch from the chemostat to the biomass recycle mode. Over 150 h, there was a two- to fourfold decrease in the respiratory potential of the microbes. After this decrease, respiratory potentials were relatively constant up to 74 days of operation. A decline in reactivity was also indicated by increasing lag periods before growth in response to organic nutrient inputs and by a decrease in the proportion of cells able to reduce tetrazolium dye. However, the bioreactor system was still capable of rapidly metabolizing inputs of organic matter, because of the very high biomass concentrations. It appears that < 10% of the organic carbon inputs accumulate as biomass.     

Response of bacterial community during bioremediation of an oil-polluted soil

AIM: To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. METHODS AND RESULTS: The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. CONCLUSIONS: The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.     

Soil Microbial Community Response to Land Use Change in an Agricultural Landscape of Western Kenya

Tropical agroecosystems are subject to degradation processes such as losses in soil carbon, nutrient depletion, and reduced water holding capacity that occur rapidly resulting in a reduction in soil fertility that can be difficult to reverse. In this research, a polyphasic methodology has been used to investigate changes in microbial community structure and function in a series of tropical soils in western Kenya. These soils have different land usage with both wooded and agricultural soils at Kakamega and Ochinga, whereas at Ochinga, Leuro, Teso, and Ugunja a replicated field experiment compared traditional continuous maize cropping against an improved N-fixing fallow system. For all sites, principal component analysis of 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles revealed that soil type was the key determinant of total bacterial community structure, with secondary variation found between wooded and agricultural soils. Similarly, phospholipid fatty acid (PLFA) analysis also separated wooded from agricultural soils, primarily on the basis of higher abundance of monounsaturated fatty acids, anteiso- and iso-branched fatty acids, and methyl-branched fatty acids in the wooded soils. At Kakamega and Ochinga wooded soils had between five 5 and 10-fold higher levels of soil carbon and microbial biomass carbon than agricultural soils from the same location, whereas total enzyme activities were also lower in the agricultural sites. Soils with woody vegetation had a lower percentage of phosphatase activity and higher cellulase and chitinase activities than the agricultural soils. BIOLOG analysis showed woodland soils to have the greatest substrate diversity. Throughout the study the two functional indicators (enzyme activity and BIOLOG), however, showed lower specificity with respect to soil type and land usage than did the compositional indicators (DGGE and PLFA). In the field experiment comparing two types of maize cropping, both the maize yields and total microbial biomass were found to increase with the fallow system. Moreover, 16S rRNA gene and PLFA analyses revealed shifts in the total microbial community in response to the different management regimes, indicating that deliberate management of soils can have considerable impact on microbial community structure and function in tropical soils.     

Micronutrient and phosphorus limitation of phytoplankton abundance in Gem Lake,Sierra Nevada,California

Nutrient addition experiments conducted during the ice-free seasons of 1983 and 1984 in Gem Lake, an alpine lake in the Sierra Nevada mountains of California, indicate that algal biomass is limited by phosphorus, in combination with iron or copper. Phosphorus additions were always required to stimulate growth, but did not do so when phosphorus was the only nutrient added. Simultaneous additions of phosphorus and iron resulted in increased levels of chlorophyll, particulate carbon, particulate nitrogen and particulate phosphorus. Simultaneous additions of phosphorus and copper resulted in increases in chlorophyll, particulate nitrogen and particulate phosphorus, not in particulate carbon. Neither iron nor copper by itself stimulated growth.Particulate N : P ratios from all seasons in Gem Lake suggest that simultaneous micronutrient and phosphorus limitation exists throughout the summer, when nutrient and biomass levels remain low; limitation by phosphorus alone may appear in the fall and spring, when biomass and major ion concentrations increase dramatically.     

Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery.

The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.     

Nutrient and carbon relations in subalpine dwarf shrubs after neighbour removal or fertilization in northern Italy

Two subalpine dwarf-shrub heath communities with differing levels of soil nutrient availability were subjected to a 3-year experimental manipulation, including nutrient addition or removal of one of the two co-dominant species from each community. The main objective of our study was to assess the relative importance of interspecific competition versus nutrient limitation in relation to soil fertility. We also aimed to investigate if and to what extent current-year shoot size, leaf-based rates of net photosynthesis and foliar nutrient status accounted for the observed changes in the aboveground biomass of the shrubs. At the end of the experiment, neighbour removal increased the aboveground biomass of all shrubs, especially in the more fertile community, while fertilization did not. We concluded that: (1) competition is more effective than nutrient limitation in structuring the vegetation of subalpine heathlands; and (2) competition intensity is stronger in the more fertile community. The observed patterns of variations in aboveground biomass were not consistently related to net photosynthetic rates, size of individual shoots and foliar nutrient status. Hence, we also concluded that the growth response of dwarf shrubs to altered environmental conditions is primarily determined by developmental plasticity.     

Plant-associated fungi support bacterial resilience following water limitation

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H₂¹⁸O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H₂¹⁸O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal–bacterial synergies may support bacterial resilience to moisture limitation.Subject terms: DNA sequencing, Fungal ecology, Stable isotope analysis, Climate-change ecology, Microbial ecology