The interaction of acidophiles driving community functional responses to the re-inoculated chalcopyrite bioleaching process.

Re-inoculation was an effective way to improve bioleaching efficiency by enhancing the synergetic effects of biogenic Fe3+ coupling with S0 oxidation. However, the complex microbial interactions after re-inoculation have received far less attention, which was crucial to the bioleaching performances. Herein, the enriched ferrous oxidizers (FeO) or sulfur oxidizers (SO) were inoculated to chalcopyrite microcosm, then they were crossly re-inoculated again to characterize the interspecific interaction patterns. The results showed that the dominant species in Fe groups were Acidithiobacillus ferrooxidans, while A. thiooxidans predominated in S groups. Introducing FeO resulted in a great disturbance by shifting the community diversity and evenness significantly (p < 0.05). In comparison, the communities intensified by SO maintained the original composition and structures. Microbial networks were constructed positively and modularly. The networks intensified by FeO were less connected and complex with less nodes and edges, but showed faster responses to the re-inoculation disturbance reflected by shorter average path length. Interestingly, the genus Leptospirillum were identified as keystones in S groups, playing critical roles in iron-oxidizing with lots of sulfur oxidizers. The introduced sulfur oxidizers enhanced microbial cooperation, formed robust community with strong bio-dissolution capability, and harbored the highest bioleaching efficiency. These findings improved our understanding about the acidophiles interactions, which drive community functional responses to the re-inoculated bioleaching process.

Differential responses of stream water and bed sediment microbial communities to watershed degradation.

The importance of microbial communities in the function of lotic ecosystems is unequivocal. However, traditional watershed studies on biodiversity have mostly focused on benthic macroinvertebrates, macroalgae and fish assemblages. Here, we investigated the diversity and interaction patterns of microbial communities in water and bed sediment of streams impacted by intensive watershed activities versus streams with relatively pristine conditions via next-generation sequencing of 16S rRNA amplicons using Illumina HiSeq platform. Both water and sediment microbial communities at forested sites had higher mean alpha-diversity than developed sites. Although microbial alpha-diversity indices were generally higher in bed sediment than water, they were comparable at forested sites. In addition, losses of taxa important in nitrogen cycle were evident particularly in bed sediment of developed sites. Interactions among microorganisms visualized by microbial network were more complex at forested sites versus developed sites, with more keystone taxa predominantly from sediment. Together, these findings suggest stream water and bed sediment microbial communities may be affected by watershed disturbances in distinctive ways, and losses of important functional microbial players and keystone taxa in bed sediment may result in decline of ecosystem functions and services. Therefore, cautions should be taken when implementing remediation strategies such as sediment dredging, and reseeding contaminated sites with key microbial players may catalyze the recovery of ecosystems.

Transport and retention of Shewanella oneidensis strain MR1 in water-saturated porous media with different grain-surface properties.

Bacterium Shewanella oneidensis strain (MR1), a facultative microorganism that plays critical role in contaminant transformation and degradation, was used as an example to decipher the transport and retention of microorganisms in water-saturated porous media with different grain-surface properties and under different ionic compositions (i.e. Na+, Ca2+, and Mg2+). Dolomite and quartz sands, which contained different surface charge properties, were used as the representative minerals. Dolomite was selected because its surface charges are significantly affected by solution composition. The mobility of MR1 in the dolomite column was lower than that in the quartz column, because the lower energy barrier between MR1 and dolomite than that between MR1 and quartz, resulting in the larger retention of MR1 in the dolomite column. The breakthrough curves were well simulated by the two sites kinetic model with HYDRUS-1D. The maximum concentration of attached bacteria (Smax) were positively correlated to the ionic strength regardless of mineral types. The values of Smax were about 1.1-4.0 times larger in the MR1-dolomite system than that in the MR1-quartz system under different ionic strength conditions. The retention of the MR1 on dolomite surfaces in the presence of divalent cations Ca2+ is significantly higher than that on quartz surfaces primarily due to the larger electrostatic attraction energy between the MR1 and dolomite grains. The findings demonstrate that the porous media with the lower negative charge has the higher capacity for the retention and deposition of MR1, potentially affecting the transport of MR1 and other bacteria in the subsurface.

Profiling microbial communities in a watershed undergoing intensive anthropogenic activities.

In lotic ecosystems highly susceptible to anthropogenic activities, the influences of environmental variables on microbial communities and their functions remain poorly understood, despite our rapidly increasing sequencing ability. In this study, we profiled the microbial communities in the hyporheic sediments of a watershed undergoing intensive anthropogenic activities via next-generation sequencing of 16S rRNA V4-V5 hypervariable regions on Illumina MiSeq platform. Tidal impacts on microbial communities were investigated via co-occurrence networks. In addition, the influences of physicochemical variables including salinity, and the concentrations of nutrients, organic matter and heavy metals on the microbial communities were explored via canonical correspondence analyses. The sediment samples were collected from 19 sites covering the whole main river stem of the target watershed (n = 19; Maozhou river watershed, Shenzhen, China). The samples were sub-divided in the field for microbiological analyses and measurements of physicochemical variables. The results indicated that core microbiome was associated with archaea methanogens and bacteria members from Proteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, Synergistetes and Firmicutes, among which, gram-negative and anaerobic bacteria genera contributing to the cycling of carbon, nitrogen and sulfur were predominant. Site-specific microbiomes were revealed that may serve as indicators of local environmental conditions (e.g., members affiliated to Oceanospirillales were abundant at sites with salt intrusion). Distinct microbial co-occurrence networks were identified for non-tidal, inter-tidal and tidal sites. Major environmental factors influencing microbial community composition included the concentrations of nitrate and bicarbonate in river water, pore water concentrations of sulfate, dissolved organic carbon and electrical conductivity, as well as manganese concentrations associated with the solid sediment. Collectively, the results of this study provide fundamental insights into the influence of environmental perturbations on microbial community composition in a lotic system, which may aid in the design of effective remediation and/or restoration strategies in the target watershed and beyond.

Interactive effects of water quality, physical habitat, and watershed anthropogenic activities on stream ecosystem health.

Ecological degradation of streams remains a major environmental concern worldwide. While stream restoration has received considerable attention, mitigation efforts focused on the improvement of physical habitat have not proven completely effective. Several small-scale studies have emphasized that effective restoration strategies require a more holistic understanding of the variables at play, although the generalization of the findings based on the small-scale studies remains unclear. Using a comprehensive statewide stream monitoring database from West Virginia (WV), a detailed landscape dataset, and a machine learning algorithm, this study explores the interactive impacts of water quality and physical habitat on stream ecosystem health as indicated by benthic macroinvertebrate scores. Given the long history of energy extraction in this region (i.e., coal mining and oil/gas production), investigation of energy extraction influences is highlighted. Our results demonstrate that a combination of good habitat and low specific conductance is generally associated with favorable benthic macroinvertebrate scores, whereas poor habitat combined with water quality conditions typically indicative of high ionic strength are associated with impaired stream status. In addition, streams impacted by both energy extraction and residential development had a higher percentage of impairment compared to those impacted predominantly by energy extraction or residential development alone. While water quality played a more important role in the ecosystem health of streams impacted primarily by energy extraction activities, habitat seems to be more influential in streams impacted by residential development. Together, these findings emphasize that stream restoration strategies should consider interactive effects of multiple environmental stressors tailored to specific sites or site types - as opposed to considering a single stressor or multiple stressors separately.

Coupling a continuous watershed-scale microbial fate and transport model with a stochastic dose-response model to estimate risk of illness in an urban watershed.

Within the United States, elevated levels of fecal indicator bacteria (FIB) remain the leading cause of surface water-quality impairments requiring formal remediation plans under the federal Clean Water Act's Total Maximum Daily Load (TMDL) program. The sufficiency of compliance with numerical FIB criteria as the targeted endpoint of TMDL remediation plans may be questionable given poor correlations between FIB and pathogenic microorganisms and varying degrees of risk associated with exposure to different fecal pollution sources (e.g. human vs animal). The present study linked a watershed-scale FIB fate and transport model with a dose-response model to continuously predict human health risks via quantitative microbial risk assessment (QMRA), for comparison to regulatory benchmarks. This process permitted comparison of risks associated with different fecal pollution sources in an impaired urban watershed in order to identify remediation priorities. Results indicate that total human illness risks were consistently higher than the regulatory benchmark of 36 illnesses/1000 people for the study watershed, even when the predicted FIB levels were in compliance with the Escherichia coli geometric mean standard of 126CFU/100mL. Sanitary sewer overflows were associated with the greatest risk of illness. This is of particular concern, given increasing indications that sewer leakage is ubiquitous in urban areas, yet not typically fully accounted for during TMDL development. Uncertainty analysis suggested the accuracy of risk estimates would be improved by more detailed knowledge of site-specific pathogen presence and densities. While previous applications of the QMRA process to impaired waterways have mostly focused on single storm events or hypothetical situations, the continuous modeling framework presented in this study could be integrated into long-term water quality management planning, especially the United States' TMDL program, providing greater clarity to watershed stakeholders and decision-makers.

Long-Term Impacts of Bacteria-Sediment Interactions in Watershed-Scale Microbial Fate and Transport Modeling.

Elevated levels of fecal indicator bacteria (FIB) remain the leading cause of surface water-quality impairments in the United States. Under the Clean Water Act, basin-specific total maximum daily load (TMDL) restoration plans are responsible for bringing identified water impairments in compliance with applicable standards. Watershed-scale model predictions of FIB concentrations that facilitate the development of TMDLs are associated with considerable uncertainty. An increasingly cited criticism of existing modeling practice is the common strategy that assumes bacteria behave similarly to "free-phase" contaminants, although many field evidence indicates a nontrivial number of cells preferentially associate with particulates. Few attempts have been made to evaluate the impacts of sediment on the predictions of in-stream FIB concentrations at the watershed scale, with limited observational data available for model development, calibration, and validation. This study evaluates the impacts of bacteria-sediment interactions in a continuous, watershed-scale model widely used in TMDL development. In addition to observed FIB concentrations in the water column, streambed sediment-associated FIB concentrations were available for model calibration. While improved model performance was achieved compared with previous studies, model performance under a "sediment-attached" scenario was essentially equivalent to the simpler "free-phase" scenario. Watershed-specific characteristics (e.g., steep slope, high imperviousness) likely contributed to the dominance of wet-weather pollutant loading in the water column, which may have obscured sediment impacts. As adding a module accounting for bacteria-sediment interactions would increase the model complexity considerably, site evaluation preceding modeling efforts is needed to determine whether the additional model complexity and effort associated with partitioning phases of FIB is sufficiently offset by gains in predictive capacity.

Storm loads of culturable and molecular fecal indicators in an inland urban stream.

Elevated concentrations of fecal indicator bacteria in receiving waters during wet-weather flows are a considerable public health concern that is likely to be exacerbated by future climate change and urbanization. Knowledge of factors driving the fate and transport of fecal indicator bacteria in stormwater is limited, and even less is known about molecular fecal indicators, which may eventually supplant traditional culturable indicators. In this study, concentrations and loading rates of both culturable and molecular fecal indicators were quantified throughout six storm events in an instrumented inland urban stream. While both concentrations and loading rates of each fecal indicator increased rapidly during the rising limb of the storm hydrographs, it is the loading rates rather than instantaneous concentrations that provide a better estimate of transport through the stream during the entire storm. Concentrations of general fecal indicators (both culturable and molecular) correlated most highly with each other during storm events but not with the human-associated HF183 Bacteroides marker. Event loads of general fecal indicators most strongly correlated with total runoff volume, maximum discharge, and maximum turbidity, while event loads of HF183 most strongly correlated with the time to peak flow in a hydrograph. These observations suggest that collection of multiple samples during a storm event is critical for accurate predictions of fecal indicator loading rates and total loads during wet-weather flows, which are required for effective watershed management. In addition, existing predictive models based on general fecal indicators may not be sufficient to predict source-specific genetic markers of fecal contamination.

Hydrometeorological and physicochemical drivers of fecal indicator bacteria in urban stream bottom sediments.

High levels of fecal indicator bacteria (FIB) are the leading cause of surface water quality impairments in the United States. Watershed-scale models are commonly used to identify relative contributions of watershed sources and to evaluate the effectiveness of remediation strategies. However, most existing models simplify FIB transport behavior as equivalent to that of dissolved-phase contaminants, ignoring the impacts of sediment on the fate and transport of FIB. Implementation of sediment-related processes within existing models is limited by minimal available monitoring data on sediment FIB concentrations for model development, calibration, and validation purposes. The purpose of the present study is to evaluate FIB levels in the streambed sediments as compared to those in the water column and to identify environmental variables that influence water and underlying sediment FIB levels. Concentrations of and enterococci in the water column and sediments of an urban stream were monitored weekly for 1 yr and correlated with a variety of potential hydrometeorological and physicochemical variables. Increased FIB concentrations in both the water column and sediments were most strongly correlated with increased antecedent 24-h rainfall, increased stream water temperature, decreased dissolved oxygen, and decreased specific conductivity. These observations will support future efforts to incorporate sediment-related processes in existing models through the identification of key FIB relationships with other model inputs, and the provision of sediment FIB concentrations for direct model calibration. In addition, identified key variables can be used in quick evaluation of the effectiveness of potential remediation strategies.

One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP.

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²âº was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²âº at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k (cat) and K(m) values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t(1/2) = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose.

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.

Insights into two high homogenous genes involved in copper homeostasis in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans, an important microorganism in bioleaching industry, has been sequenced recently, and from the annotated information, there are four genes involved in copper homeostasis. Sequence analysis showed that two of them, Afe0329 and Afe0663, were high homologous (94.43% identity). With the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) cloning approach, the differential gene expression of these two high homologous genes in a genome was successfully identified for the first time. In comparison with Afe0663, Afe0329 was highly expressed grown in the medium with copper, and the restriction fragment length polymorphism (RFLP) profile showed that 96% of lanes were products of Afe0329. Analysis of the protein sequence encoded by Afe0329 suggested a conserved domain of P1b3-type ATPase, which is a heavy-metal pump, and, to be unexpected, the molecular modeling revealed that the amino acids determining the type of heavy-metal pumps were responsible for the gate of the copper ion channel in the transmembrane area of the protein. The activity of P1b-type ATPase disrupted in Escherichia coli could be partially rescued by complementation by the plasmid-carrying Afe0329 gene. All of these results suggest that a copper homeostasis mechanism including P-type ATPase is of importance for the survival of this extremophilic microorganism.