A direct contact bioassay using immobilized microalgal balls to evaluate the toxicity of contaminated field soils.

The present study used a bioassay of immobilized microalgae (Chlorella vulgaris) via direct contact to assess the toxicity of eleven uncontaminated (reference) and five field contaminated soils with various physicochemical properties and contamination. Photosynthetic oxygen concentration in the headspace of the test kit by Chlorella vulgaris in the reference soils ranged between 12.93% and 14.80% and only 2.54%-7.14% in the contaminated soils, respectively. Inherent test variability (CVi) values ranged between 2.90% and 9.04%; variation due to soil natural properties (CVrs) ranged between 0.33% and 13.0%; and minimal detectable difference (MDD) values ranged from 4.69% to 11.6%. A computed toxicity threshold of 15% was established for microalgae soil toxicity tests based on calculations of the maximal tolerable inhibition (MTI). All contaminated soils were considered toxic to microalgae because their levels of inhibition ranged between 39.5% and 82.9%, exceeding the 15% toxicity threshold. It can be concluded that the elevated concentrations of heavy metals and organic contaminants in the contaminated soils induced the higher inhibitory levels. Overall, direct contact soil toxicity tests using immobilized microalgae provided coherent and repeatable data and can be utilized as a simple and suitable tool for the toxicity testing of contaminated field soils.

Enhancing anaerobic digestion for rural wastewater treatment with granular activated carbon (GAC) supplementation.

Notwithstanding many efforts to increase the efficiency of anaerobic digestion at low-temperature (winter) conditions, a cost-effective and efficient method is lacking. This study proposes a low-cost method of low-temperature (<35 °C) anaerobic digestion of wastewater, involving supplementation with granular activated carbon (GAC). Supplementation with GAC was found to reduce the lag time by 29.8% (from 15.1 to 10.6 days) and increase the maximum methane production rate by 23.4% (from 6.4 to 7.9 mL/day) at 25 °C. Network analysis demonstrated a strong co-occurrence of Syntrophobacteriales and hydrogenotrophic methanogens (Methanobacteriaceae; WSA2; Methanoregulaceae). GAC supplementation can drastically reduce the time required for organic matter decomposition and methane production, thereby increase the efficiency of wastewater treatment.

Effects of wire-type and mesh-type anode current collectors on performance and electrochemistry of microbial fuel cells.

Carbon-based material is commonly used for anodes in MFCs, but its low conductivity often limits anodic performance. Application of corrosion-resistive current collector to carbon-based anode can be a promising strategy for increasing the anodic performance. In this study, it was hypothesized increasing metal current collector improved anodic performance. Two different carbon-felt anodes with titanium wires (CF-W) or stainless steel mesh (CF-M) as a current collector were tested in a single chamber MFC. In the short-term tests such as polarization and impedance tests, CF-M with the larger current collector area (21.7 cm2) had 33% higher maximum power (2311 mW/m2), 81% lower anodic resistance (3 Ω), and 92% lower anodic impedance (1.1 Ω). However, in the long-term tests, CF-W with the smaller current collector area (0.6 cm2) showed higher performance in power and current generation, COD removal, and CE (51%, 10%, 11%, and 5% higher, respectively) and produced 41% higher net current in cyclic voltagramm (20.0 mA vs. 14.2 mA). This result shows that larger current collector is advantageous in short-term performance and disadvantageous in long-term performance, because the larger current collector is good for current collection, but interferes with mass transfer and microbial growth.

Polymer Film-Based Screening and Isolation of Polylactic Acid (PLA)-Degrading Microorganisms.

Polylactic acid (PLA) has been highlighted as an alternative renewable polymer for the replacement of petroleum-based plastic materials, and is considered to be biodegradable. On the other hand, the biodegradation of PLA by terminal degraders, such as microorganisms, requires a lengthy period in the natural environment, and its mechanism is not completely understood. PLA biodegradation studies have been conducted using mainly undefined mixed cultures, but only a few bacterial strains have been isolated and examined. For further characterization of PLA biodegradation, in this study, the PLA-degrading bacteria from digester sludge were isolated and identified using a polymer film-based screening method. The enrichment of sludge on PLA granules was conducted with the serial transference of a subculture into fresh media for 40 days, and the attached biofilm was inoculated on a PLA film on an agar plate. 3D optical microscopy showed that the isolates physically degraded the PLA film due to bacterial degradation. 16S rRNA gene sequencing identified the microbial colonies to be Pseudomonas sp. MYK1 and Bacillus sp. MYK2. The two isolates exhibited significantly higher specific gas production rates from PLA biodegradation compared with that of the initial sludge inoculum.

Enhanced biomass production through optimization of carbon source and utilization of wastewater as a nutrient source.

The study aimed to utilize the domestic wastewater as nutrient feedstock for mixotrophic cultivation of microalgae by evaluating appropriate carbon source. The microalgae Chlorella vulgaris was cultivated in municipal wastewater under various carbon sources (glucose, glycerol, and acetate), followed by optimization of appropriate carbon source concentration to augment the biomass, lipid, and carbohydrate contents. Under optimized conditions, namely of 5 g/L glucose, C. vulgaris showed higher increments of biomass with 1.39 g/L dry cell weight achieving biomass productivity of 0.13 g/L/d. The biomass accumulated 19.29 ± 1.83% total lipid, 41.4 ± 1.46% carbohydrate, and 33.06 ± 1.87% proteins. Moreover, the cultivation of Chlorella sp. in glucose-supplemented wastewater removed 96.9% chemical oxygen demand, 65.3% total nitrogen, and 71.2% total phosphate. The fatty acid methyl ester obtained showed higher amount (61.94%) of saturated fatty acid methyl esters associated with the improved fuel properties. These results suggest that mixotrophic cultivation using glucose offers great potential in the production of renewable biomass, wastewater treatment, and consequent production of high-value microalgal oil.

Assessment of microbial diversity bias associated with soil heterogeneity and sequencing resolution in pyrosequencing analyses.

It is important to estimate the true microbial diversities accurately for a comparative microbial diversity analysis among various ecological settings in ecological models. Despite drastically increasing amounts of 16S rRNA gene targeting pyrosequencing data, sampling and data interpretation for comparative analysis have not yet been standardized. For more accurate bacterial diversity analyses, the influences of soil heterogeneity and sequence resolution on bacterial diversity estimates were investigated using pyrosequencing data of oak and pine forest soils with focus on the bacterial 16SrRNA gene. Soil bacterial community sets were phylogenetically clustered into two separate groups by forest type. Rarefaction curves showed that bacterial communities sequenced from the DNA mixtures and the DNAs of the soil mixtures had midsize richness compared with other samples. Richness and diversity estimates were highly variable depending on the sequence read numbers. Bacterial richness estimates (ACE, Chao 1 and Jack) of the forest soils had positive linear relationships with the sequence read number. Bacterial diversity estimates (NPShannon, Shannon and the inverse Simpson) of the forest soils were also positively correlated with the sequence read number. One-way ANOVA shows that sequence resolution significantly affected the α-diversity indices (P<0.05), but the soil heterogeneity did not (P>0.05). For an unbiased evaluation, richness and diversity estimates should be calculated and compared from subsets of the same size.

Performance and bacterial communities of successive alkalinity-producing systems (SAPSs) in passive treatment processes treating mine drainages differing in acidity and metal levels.

Successive alkalinity-producing systems (SAPSs) is a key unit process in the passive treatment of acidic mine drainage. Physico-chemistry and pyrosequencing-based bacterial communities of two passive treatment processes in Gapjung (GJ) and Seokbong (SB) were analyzed. The influent of SB harbored higher levels of acidity and metals than that of GJ. SAPS-SB demonstrated better performance of acidity neutralization and metal removal than SAPS-GJ, despite its shorter hydraulic retention time and higher acidity. System diagnosis revealed that the capacities of SAPSs were not well predicted in the design steps. Bacterial diversity indices and composition were compared at the same sequence read number for fair evaluation. Most of the bacterial sequences were affiliated with uncultured species. A notable difference was observed in the bacterial community compositions of the SAPSs in GJ and SB. Classes of putative sulfate-reducing bacteria, Clostridia (8.3 %) and Deltaproteobacteria (6.1 %), were detected in SAPS-GJ, and Clostridia (14.6 %) was detected in SAPS-SB. Bacilli, which is not a known sulfate-reducing bacterial group, was the second largest class (12.8 %) in SAPS-GJ and the largest class (51.1 %) in SAPS-SB, suggesting that Bacilli may have a prominent role in SAPS. One hundred ninety operational taxonomic units were shared, which occupied ~10 % of each number of total operational taxonomic units in SAPS-GJ and SAPS-SB, respectively. Bacilli and Clostridia were the major shared classes, and Bacillus, Lysinibacillus, and Ureibacillus were the major shared genera. Rarefaction analysis, richness estimates, diversity estimates, and abundance rank analysis show that the sediment bacterial community of SAPS-GJ was more diverse and more evenly distributed than that of SAPS-SB.

Nitrate-contaminated groundwater remediation by combined autotrophic and heterotrophic denitrification for sulfate and pH control: batch tests.

Groundwater remediation was evaluated for combined autotrophic and heterotrophic denitrification under high (154 mg/L as CaCO3) and low (95 mg/L as CaCO3) alkaline conditions. Two levels of acetate (47 and 94 mg/L) and ethanol (24 and 48 mg/L) were added to the reactors. Obtained denitrification rates were 2.89, 2.58, 3.55, 1.96, and 2.0 mg-N/L · h for high alkaline conditions, whereas under low alkaline conditions has given 2.36, 1.94, 2.47, 2.74, and 2.29 mg-N/L · h for control, 47 and 94 mg/L acetate, and 24 and 48 mg/L ethanol, respectively. Nitrite was accumulated for controls but reactors with acetate and ethanol did not accumulate nitrite. Acetate and ethanol addition decreased sulfate to nitrate ratios in the range of 4.5-7.58 for high alkaline conditions (12.77 for control) and 4.43-6.78 for low alkaline conditions (7.90 for control). Acetate was more efficient compared with ethanol in controlling sulfate production and pH maintenance.

Effects of substrate concentrations on performance of serially connected microbial fuel cells (MFCs) operated in a continuous mode.

Stacking of microbial fuel cells (MFC) by connecting multiple small-sized units in a series is used for generating higher power from the MFCs. However, voltage reversal is a critical problem in a serially connected MFC unit. The voltage reversal often occurs when substrate concentration is relatively low in the anodic compartment. Two rectangular individual cells were stacked together in series: MFC1 was fed with 1 g glucose L(-1) throughout the experiment while MFC2 was fed with various concentrations of glucose (0.1, 0.2, 0.3, 0.5 and 0.8 g L(-1)). Voltage reversal occurred when the stack configuration was performed using (1 + 0.1) g glucose L(-1). The stacked configurations with (1 + 0.2, 1 + 0.3, 1 + 0.5 and 1 + 0.8) g glucose L(-1) were operated successfully without the voltage reversal. The maximum powers of 1.88, 2.04, 3.6, 2.5 and 2.18 mW were obtained with the stacked configurations of (1 + 0.2), (1 + 0.3), (1 + 0.5), (1 + 0.8) and (1 + 1) g glucose L(-1), respectively. Except in the stacked configuration with (1 + 0.1) g glucose L(-1), the stacked voltages obtained were similar.

Impedance characteristics and polarization behavior of a microbial fuel cell in response to short-term changes in medium pH.

pH oppositely influences anode and cathode performance in microbial fuel cells. The differential electrochemical effects at each electrode and the resultant full-cell performance were analyzed in medium pH from 6.0 to 8.0. Potentials changed -60 mV/pH for the anode and -68 mV/pH for the cathode, coincident with thermodynamic estimations. Open circuit voltage reached a maximum (741 mV) at pH 7, and maximum power density was highest (712 mW/m²) at pH 6.5 as the cathode performance improved at lower pH. Maximum current density increased and apparent half-saturation potential (E(KA)) decreased with increasing medium pH due to improved anode performance. An equivalent circuit model composed of two time constant processes accurately fit bioanode impedance data. One of these processes was consistently the rate-limiting step for acetate-oxidizing exoelectrogenesis, with its pH-varying charge transfer resistance R2 ranging from 2- to 321-fold higher than the pH-independent charge transfer resistance R1. The associated capacitance C2 was 2-3 orders of magnitude larger than C1. R2 was lowest near E(KA) and increased by several orders of magnitude at anode potentials above E(KA), while R1 was nearly stable. However, fits deviated slightly at potentials above E(KA) due to emerging impedance possibly associated with diffusion and excessive potential.

Influence of external resistance on electrogenesis, methanogenesis, and anode prokaryotic communities in microbial fuel cells.

The external resistance (R(ext)) of microbial fuel cells (MFCs) regulates both the anode availability as an electron acceptor and the electron flux through the circuit. We evaluated the effects of R(ext) on MFCs using acetate or glucose. The average current densities (I) ranged from 40.5 mA/m(2) (9,800 Ω) to 284.5 mA/m(2) (150 Ω) for acetate-fed MFCs (acetate-fed reactors [ARs]), with a corresponding anode potential (E(an)) range of -188 to -4 mV (versus a standard hydrogen electrode [SHE]). For glucose-fed MFCs (glucose-fed reactors [GRs]), I ranged from 40.0 mA/m(2) (9,800 Ω) to 273.0 mA/m(2) (150 Ω), with a corresponding E(an) range of -189 to -7 mV. ARs produced higher Coulombic efficiencies and energy efficiencies than GRs over all tested R(ext) levels because of electron and potential losses from glucose fermentation. Biogas production accounted for 14 to 18% of electron flux in GRs but only 0 to 6% of that in ARs. GRs produced similar levels of methane, regardless of the R(ext). However, total methane production in ARs increased as R(ext) increased, suggesting that E(an) might influence the competition for substrates between exoelectrogens and methanogens in ARs. An increase of R(ext) to 9,800 Ω significantly changed the anode bacterial communities for both ARs and GRs, while operating at 970 Ω and 150 Ω had little effect. Deltaproteobacteria and Bacteroidetes were the major groups found in anode communities in ARs and GRs. Betaproteobacteria and Gammaproteobacteria were found only in ARs. Bacilli were abundant only in GRs. The anode-methanogenic communities were dominated by Methanosaetaceae, with significantly lower numbers of Methanomicrobiales. These results show that R(ext) affects not only the E(an) and current generation but also the anode biofilm community and methanogenesis.

Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors.

Microbial fuel cells (MFCs) harness the electrochemical activity of certain microbes for the production of electricity from reduced compounds. Characterizations of MFC anode biofilms have collectively shown very diverse microbial communities, raising ecological questions about competition and community succession within these anode-reducing communities. Three sets of triplicate, two-chamber MFCs inoculated with anaerobic sludge and differing in energy sources (acetate, lactate, and glucose) were operated to explore these questions. Based on 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE), all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone in the Bacteroidetes class (99% similarity). Various other Geobacter-like sequences were also enriched in most of the anode biofilms. While the anode communities in replicate reactors for each substrate generally converged to a reproducible community, there were some variations in the relative distribution of these putative anode-reducing Geobacter-like strains. Firmicutes were found only in glucose-fed MFCs, presumably serving the roles of converting complex carbon into simple molecules and scavenging oxygen. The maximum current density in these systems was negatively correlated with internal resistance variations among replicate reactors and, likely, was only minimally affected by anode community differences in these two-chamber MFCs with high internal resistance.