Enhanced bioremediation of acid mine-influenced groundwater with micro-sized emulsified corn oil droplets (MOD) and sulfate-reducing bacteria (Desulfovibrio vulgaris) in a microcosm assay.

High concentrations of metals and sulfates in acid mine drainage (AMD) are the cause of the severe environmental hazard that mining operations pose to the surrounding ecosystem. Little study has been conducted on the cost-effective biological process for treating high AMD. The current research investigated the potential of the proposed carbon source and sulfate reduction bacteria (SRB) culture in achieving the bioremediation of sulfate and heavy metals. This work uses individual and combinatorial bioaugmentation and bio-stimulation methods to bioremediate acid-mine-influenced groundwater in batch microcosm experiments. Bioaugmentation and bio-stimulation methods included pure culture SRB (Desulfovibrio vulgaris) and microsized oil droplet (MOD) by emulsifying corn oil. The research tested natural attenuation (T 1), bioaugmentation (T2), biostimulation (T3), and bioaugmentation plus biostimulation (T4) for AM-contaminated groundwater remediation. Bioaugmentation and bio-stimulation showed the greatest sulfate reduction (75.3%) and metal removal (95-99%). Due to carbon supply scarcity, T1 and T2 demonstrated 15.7% and 27.8% sulfate reduction activities. Acetate concentrations in T3 and T4 increased bacterial activity by providing carbon sources. Metal bio-precipitation was substantially linked with sulfate reduction and cell growth. SEM-EDS study of precipitates in T3 and T4 microcosm spectra indicated peaks for S, Cd, Mn, Cu, Zn, and Fe, indicating metal-sulfide association for metal removal precipitates. The MOD provided a constant carbon source for indigenous bacteria, while Desulfovibrio vulgaris increased biogenic sulfide synthesis for heavy metal removal.

Establishment of wastewater-based SARS-CoV-2 monitoring system over two years: Case studies in South Korea.

With the global COVID-19 pandemic, wastewater surveillance has received a considerable attention as a method for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater treatment plant (WWTP) and sewer systems. For the first time in Korea, this study utilized the wastewater surveillance technique to monitor the COVID-19 outbreak. Sampling efforts were carried out at the WWTPs in the capital city of Korea, Seoul, and Daegu the place where the first severe outbreak was reported. The RNA of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been extracted from the collected wastewater influent and primary sewage sludge samples. The outcomes were contrasted with the COVID-19 cases in the WWTPs served area. Additionally, whole transcriptome sequencing was used to compare the microbial community alterations before and after the COVID-19 outbreak and SARS-CoV-2 variations. The results demonstrated that the changes in SARS-CoV-2 RNA concentrations in the influent and sludge matched the trends of reported COVID-19 cases, especially sludge showed high-resolution data, which is well-matched when fewer COVID-19 cases (0-250) are reported. Interestingly, one month before the clinical report, we found that the SARS-CoV-2 Beta variant (South Africa, B.1.351) in the wastewater. In addition, the Aeromonas bacterial species was dominated (21.2%) among other bacterial species in wastewater after the COVID-19 outbreak, suggesting a potential indirect microbial indicator of the COVID-19 outbreak.

Seasonal trends of mercury bioaccumulation and assessment of toxic effects in Asian clams and microbial community from field study of estuarine sediment.

This study investigated seasonal trends in bioaccumulation potential and toxic effects of mercury (Hg) in Asian clams (Corbicula fluminea) and microbial community. For this, a clam-exposure experiment was performed during summer, fall, and winter seasons in four different sites (HS1: control/clean site; HS2, HS3, and HS4: contaminated sites) of Hyeongsan River estuary, South Korea. Total mercury (THg) and methylmercury (MeHg) in whole sediments were highest at HS4 site during fall, sustained similar levels during winter, but decreased during summer. Unlike whole sediment, pore water reported higher levels in summer, and gradually declined during fall and winter. Asian clams from HS4 site collected during summer presented highest bioaccumulations of THg (521.52 µg/kg, dry weight) and MeHg (161.04 µg/kg, dry weight), which also correlated with the higher levels of Hg present in pore water in the same season. Moreover, biota-sediment-pore water accumulation factor (BSpAF) were comparatively greater in clams collected from HS2∼HS4 compared to HS1 sites, suggesting that porewater was a better indicator of accumulation of Hg. Upregulation of biomarker genes responsible for detoxifying process (gsts1), scavenging oxidative stress (cat), and protein reparation (hsp70 and hsp90) were observed in clams collected from HS2∼HS4. The overexpression of these biomarkers implied that Asian clams can be considered as promising warning tools for Hg-contamination. Both bacterial and metabolic diversities were negatively affected by higher levels of THg and MeHg. Phylum Proteobacteria was enriched in HS2∼HS4 compared to HS1. In contrast, phylum Bacteroidetes showed a reverse trend. The metabolic profile was highest in HS1 and lowest in HS4, revealing higher stress of Hg in HS4 site. Overall, the outcomes of this field study broaden the information on seasonal trends of bioaccumulation of Hg and its toxic effects. These findings may be helpful in Hg monitoring and management programs in other river systems.

Evaluation of DNA extraction yield from a chlorinated drinking water distribution system.

Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 µm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 102-1.5 × 103 cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E. coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

Effect of organic micropollutants on biofouling in a forward osmosis process integrating seawater desalination and wastewater reclamation.

This study systematically investigated the effect of organic micropollutants (OMPs) on biofouling in forward osmosis (FO) integrating wastewater treatment and seawater dilution. Synthetic seawater (0.6 M sodium chloride) was used as a draw solution and synthetic municipal wastewater as a feed solution. To evaluate the impact of OMPs in a replicate parallel study, wastewater was supplemented with a mixture of 7 OMPs (OMPs-feed) and without OMPs (control) during 8 batch filtration cycles with feed and draw solution replacement after each filtration. The FO performance (water flux), development and microbial composition properties of biofilm layers on the wastewater side of the FO membrane were studied. Compared to the control without OMPs, the FO fed with OMPs containing wastewater showed (i) initially the same water flux and flux decline during the first filtration cycle, (ii) with increasing filtration cycle a lower flux decline and (iii) lower concentrations for the total cells, ATP, EPS carbohydrates and proteins in biofilm layers, and (iv) a lower diversity of the biofilm microbial community composition (indicating selective pressure) and (v) increasing rejection of 6 of the 7 OMPs. In essence, biofouling on the FO membrane showed (i) a lower flux decline in the presence of OMPs in the feed water and (ii) a higher OMPs rejection, both illustrating better membrane performance. This study has a significant implication for optimizing osmotic dilution in terms of FO operation and OMPs rejection.

Effect of phosphate availability on biofilm formation in cooling towers.

Phosphate limitation has been suggested as a preventive method against biofilms. P-limited feed water was studied as a preventive strategy against biofouling in cooling towers (CTs). Three pilot-scale open recirculating CTs were operated in parallel for five weeks. RO permeate was fed to the CTs (1) without supplementation (reference), (2) with supplementation by biodegradable carbon (P-limited) and (3) with supplementation of all nutrients (non-P-limited). The P-limited water contained ≤10 µg PO4 l-1. Investigating the CT-basins and coupons showed that P-limited water (1) did not prevent biofilm formation and (2) resulted in a higher volume of organic matter per unit of active biomass compared with the other CTs. Exposure to external conditions and cycle of concentration were likely factors that allowed a P concentration sufficient to cause extensive biofouling despite being the limiting compound. In conclusion, phosphate limitation in cooling water is not a suitable strategy for CT biofouling control.

Insignificant Impact of Chemotactic Responses of Pseudomonas aeruginosa on the Bacterial Attachment to Organic Pre-Conditioned RO Membranes.

We investigated the impact of conditioning compositions on the way bacteria move and adhere to reverse osmosis (RO) membranes that have been pre-conditioned by organic compounds. We used humic acid (HA), bovine serum albumin (BSA), and sodium alginate (SA) to simulate conditioning layers on the RO membranes. First, we investigated the chemotactic responses of Pseudomonas aeruginosa PAO1 to the organic substances and the impact of changes in physicochemical characteristics of pre-conditioned membranes on bacterial attachment. Second, we observed bacterial attachment under the presence or absence of nutrients or microbial metabolic activity. Results showed that there was no relationship between the chemotactic response of P. aeruginosa PAO1 and the organic substances, and the changes in hydrophobicity, surface free energy, and surface charge resulting from changing the composition of the conditioning layer did not seem to affect bacterial attachment, whereas changing the roughness of the conditioned membrane exponentially did (exponential correlation coefficient, R2 = 0.85). We found that the initial bacterial attachment on the membrane surface is influenced by (i) the nutrients in the feed solution and (ii) the microbial metabolic activity, whereas the chemotaxis response has a negligible impact. This study would help to establish a suitable strategy to manage bacterial attachment.

Physiological Responses of Salinity-Stressed Vibrio sp. and the Effect on the Biofilm Formation on a Nanofiltration Membrane.

This study evaluated the effects of salinity on the physiological characteristics of Vibrio sp. B2 and biofilm formation on nanofiltration (NF) membrane coupons used in the high recovery seawater desalination process. The test conditions were at 0.6, 1.2, and 2.4 M sodium chloride (NaCl), equivalent to salinity of seawater, brine at 50% and 75% water recovery, respectively. High salinity inhibited the cell growth rate but increased the viability and bacterial membrane integrity. In addition, protein and eDNA concentrations of salinity-stressed bacteria were increased at 1.2 and 2.4 M NaCl. In particular, protein concentration was linearly correlated with the NaCl concentration. Similarly, less biofilm formation on the NF membrane coupon (without permeation flux) was observed by the salinity-stressed bacteria; however, the production of extracellular polymeric substances (EPS) was significantly increased as compared to control, and protein was an influential factor for biofilm formation. This study shows that salinity-stressed bacteria have a high potential to cause biofouling on membrane surface as the bacteria still maintain the cell activity and overproduce EPS. The potential of biofilm formation by the salinity-stressed bacteria has not been reported. Therefore, the findings are important to understand the mechanisms of membrane biofouling in a high salinity environment.

Cleaning efficacy of hydroxypropyl-beta-cyclodextrin for biofouling reduction on reverse osmosis membranes.

In this study, an environmentally friendly compound, hydroxypropyl-beta-cyclodextrin (HP-ß-CD) was applied to clean reverse osmosis (RO) membranes fouled by microorganisms. The cleaning with HP-ß-CD removed the biofilm and resulted in a flux recovery ratio (FRR) of 102%. As cleaning efficiency is sometimes difficult to determine using flux recovery data alone, attached bacterial cells and extracellular polymeric substances (EPS) were quantified after cleaning the biofouled membrane with HP-ß-CD. Membrane surface characterization using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and atomic force microscopy (AFM) confirmed the effectiveness of HP-ß-CD in removal of biofilm from the RO membrane surface. Finally, a comparative study was performed to investigate the competitiveness of HP-ß-CD with other known cleaning agents such as sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), Tween 20, rhamnolipid, nisin, and surfactin. In all cases, HP-ß-CD was superior.

Use of rhamnolipid biosurfactant for membrane biofouling prevention and cleaning.

Rhamnolipids were evaluated as biofouling reducing agents in this study. The permeability of the bacterial outer membrane was increased by rhamnolipids while the growth rate of Pseudomonas aeruginosa was not affected. The surface hydrophobicity was increased through the release of lipopolysaccharides and extracellular polymeric substances from the outer cell membrane. Rhamnolipids were evaluated as agents for the prevention and cleaning of biofilms. A high degree of biofilm detachment was observed when the rhamnolipids were used as a cleaning agent. In addition, effective biofilm reduction occurred when rhamnolipids were applied to various species of Gram-negative bacteria isolated from seawater samples. Biofilm reduction using rhamnolipids was comparable to commercially available surfactants. In addition, 20% of the water flux was increased after rhamnolipid treatment (300 µg ml(-1), 6 h exposure time) in a dead-end filtration system. Rhamnolipids appear to have promise as biological agents for reducing membrane biofouling.

Physicochemical interactions between rhamnolipids and Pseudomonas aeruginosa biofilm layers.

This study investigated the physicochemical interactions between a rhamnolipid biosurfactant and a biofilm layer. A concentration of 300 µg mL(-1) of rhamnolipids, which is around the critical micelle concentration value (240 µg mL(-1)), showed great potential for reducing biofilm. The surface free energy between the rhamnolipids and biofilm layer decreased, as did the negative surface charge, due to the removal of negatively charged humic-like, protein-like, and fulvic acid-like substances. The carbohydrate and protein concentrations composed of extracellular polymeric substances decreased by 31.6% and 79.6%, respectively, at a rhamnolipid concentration of 300 µg mL(-1). In particular, rhamnolipids can interact with proteins, leading to a reduction of the N source and amide groups on the membrane. For carbohydrates, the component ratio of glucosamine was decreased, but the levels of glucose and mannose that form the majority of the carbohydrates remained unchanged. To our knowledge, the present study is the first attempt at studying the interactions of the two phases of rhamnolipids and the biofilm layer, and as such is expected to clarify the mechanism by which rhamnolipids lead to a reduction in biofilm.

Biofouling potential reductions using a membrane hybrid system as a pre-treatment to seawater reverse osmosis.

Biofouling on reverse osmosis (RO) membranes is the most serious problem which affects desalination process efficiency and increases operation cost. The biofouling cannot be effectively removed by the conventional pre-treatment traditionally used in desalination plants. Hybrid membrane systems coupling the adsorption and/or coagulation with low-pressure membranes can be a sustainable pre-treatment in reducing membrane fouling and at the same time improving the feed water quality to the seawater reverse osmosis. The addition of powder activated carbon (PAC) of 1.5 g/L into submerged membrane system could help to remove significant amount of both hydrophobic compounds (81.4%) and hydrophilic compounds (73.3%). When this submerged membrane adsorption hybrid system (SMAHS) was combined with FeCl(3) coagulation of 0.5 mg of Fe(3+)/L, dissolved organic carbon removal efficiency was excellent even with lower dose of PAC (0.5 g/L). Detailed microbial studies conducted with the SMAHS and the submerged membrane coagulation-adsorption hybrid system (SMCAHS) showed that these hybrid systems can significantly remove the total bacteria which contain also live cells. As a result, microbial adenosine triphosphate (ATP) as well as total ATP concentrations in treated seawater and foulants was considerably decreased. The bacteria number in feed water prior to RO reduced from 5.10E(+06) cells/mL to 3.10E(+03) cells/mL and 9.30E(+03) cells/mL after SMAHS and SMCAHS were applied as pre-treatment, respectively. These led to a significant reduction of assimilable organic carbon (AOC) by 10.1 µg/L acetate-C when SMCAHS was used as a pre-treatment after 45-h RO operation. In this study, AOC method was modified to measure the growth of bacteria in seawater by using the Pseudomonas P.60 strain.

Isolation and characterization of ethylbenzene degrading Pseudomonas putida E41.

Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (µ(max)) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h(-1), respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70-80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.

Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 µg/L, with a limit of detection of 0.03 µg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.