Isolation and screening of indigenous microalgae species for domestic and livestock wastewater treatment, biodiesel production, and carbon sequestration.

The use of indigenous microalgae strains for locally generated domestic (DWW) and livestock wastewater (LWW) treatment is essential for effective and economical applications. Phototrophic microalgae-based biofuel production also contributes to carbon sequestration via CO2 fixation. However, simultaneous consideration of both isolation and screening procedures for locally collected indigenous microalgae strains is not common in the literature. We aimed to isolate indigenous microalgae strains from locally collected samples on coastlines and islands in South Korea. Among five isolated strains, Chlorella sorokiniana JD1-1 was selected for DWW and LWW treatment due to its ability to grow in waste resources. This strain showed a higher specific growth rate in DWW than artificial growth medium (BG-11) with a range of 0.137-0.154 d-1. During cultivation, 96.5%-97.1% of total nitrogen in DWW and 89.2% in LWW was removed. Over 99% of total phosphorus in DWW and 96.4% in LWW was also removed. Finally, isolated C. sorokiniana JD1-1 was able to fix CO2 within a range of 0.0646-0.1043 g CO2 L-1 d-1. These results support the domestic applications of carbon sequestration-efficient microalgae in the waste-to-energy nexus.

Manipulation of Unfrozen Water Retention for Enhancing Petroleum Hydrocarbon Biodegradation in Seasonally Freezing and Frozen Soil.

Manipulating the retention of unfrozen water in freezing contaminated soil to achieve prolonged bioremediation in cold climates remains unformulated. This freezing-induced biodegradation experiment shows how nutrient and zeolite amendments affect unfrozen water retention and hydrocarbon biodegradation in field-aged, petroleum-contaminated soils undergoing seasonal freezing. During soil freezing at a site-specific rate (4 to -10 °C and -0.2 °C/d), the effect of nutrients was predominant during early freezing (4 to -5 °C), alleviating the abrupt soil-freezing stress near the freezing-point depressions, elevating alkB1 gene-harboring populations, and enhancing hydrocarbon biodegradation. Subsequently, the effect of increased unfrozen water retention associated with added zeolite surface areas was critical in extending hydrocarbon biodegradation to the frozen phase (-5 to -10 °C). A series of soil-freezing characteristic curves with empirical α-values (soil-freezing index) were constructed for the tested soils and shown alongside representative curves for clays to sands, indicating correlations between α-values and nutrient concentrations (soil electrical conductivity), zeolite addition (surface area), and hydrocarbon biodegradation. Heavier hydrocarbons (F3: C16-C34) notably biodegraded in all treated soils (22-37% removal), as confirmed by biomarker-based analyses (17α(H),21ß(H)-hopane), whereas lighter hydrocarbons were not biodegraded. Below 0 °C, finer-grained soils (high α-values) can be biostimulated more readily than coarser-grained soils (low α-values).

A proposed stepwise screening framework for the selection of polycyclic aromatic hydrocarbon (PAH)-degrading white rot fungi.

This study suggests a simple three-step screening protocol for the selection of white rot fungi (WRF) capable of degrading polycyclic aromatic hydrocarbons (PAHs), which combines easily applicable bioassay techniques, and verifies that protocol by evaluating the PAH degradation activity, ligninolytic enzyme secretion, and relevant gene expressions of the selected PAH-degraders. Using 120 fungal strains, a sequence of bioassay techniques was applied: Bavendamm's reaction (Step 1), remazol brilliant blue R (RBBR) decolorization (Step 2); assays for tolerance to four mixed PAHs-phenanthrene, anthracene, fluoranthene, and pyrene (Step 3). This stepwise protocol selected 14 PAH-degrading WRF, including Microporus vernicipes, Peniophora incarnata, Perenniporia subacida, Phanerochaete sordida, Phlebia acerina, and Phlebia radiata. Of these, P. incarnata exhibited the highest PAH degradative activity, ranging from 40 to > 90%, which was related to the time-variable secretions of three extracellular ligninolytic enzymes: laccase, manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP). Laccase and MnP production by P. incarnata tended to be greater in the early stages of PAH degradation, whereas its LiP production became intensified with decreasing laccase and MnP production. Pilc1 and pimp1 genes encoding laccase and MnP were expressed, indicating the occurrence of extracellular enzyme-driven biodegradation of PAH by the fungal strains.

Multiple evaluation of the potential toxic effects of sediments and biota collected from an oil-polluted area around Abu Ali Island, Saudi Arabia, Arabian Gulf.

After the Gulf War Oil Spill, there have been many investigations about distributions of oil-derived pollutants nearby areas, but lacking in ecotoxicological assessment. We evaluated the potential toxicity of asphalt mats, sediments, and biota (polychaetes, chitons, snapping shrimps, and crabs) by combining two bioassays (H4IIE-luc and Vibrio fischeri) and in situ microbial community (eDNA). Samples were collected from Abu Ali Island, and organic extracts were bioassayed and further fractionated according to the chemical polarity using silica gel column. Great aryl hydrocarbon receptor (AhR)-mediated potencies and inhibition of bioluminescence were mainly found in aromatics (F2) and saturates (F1) fractions of asphalt mat and sediments, respectively, while great toxicological responses in biota samples were found in resins and polar (F3) fraction. We also confirmed that potential toxicities of biota were species-specific; great AhR-mediated potencies were found in polychaetes and great bioluminescence inhibitions were found in crabs. In microbial communities, most genera (up to 90%) were associated with polycyclic aromatic hydrocarbons (PAHs)-degrading bacteria, supporting that PAHs are the primary stressors of the benthic community around Abu Ali Island. The present study provides useful information on the contamination status, risk assessment of environmental matrices and benthic organisms in Abu Ali Island.

Echinicola sediminis sp. nov., a marine bacterium isolated from coastal sediment.

A novel Gram-stain-negative, rod-shaped, gliding bacterium, designated strain 001-Na2T, was isolated from coastal sediment of the Yellow Sea in Sindu-ri, Republic of Korea. Cells were aerobic, oxidase-positive and catalase-positive. Colonies were dark salmon-coloured, circular, and pulvinate with entire margins. 16S rRNA gene sequence analysis revealed that strain 001-Na2T belonged to the genus Echinicola and these species formed a distinct lineage within the family Cyclobacteriaceae. The closest phylogenetic neighbours were 'Echinicola shivajiensis' AK12T (98.1 % 16S rRNA gene sequence similarity), Echinicola vietnamensis LMG 23754T (96.4 %), Echinicola jeungdonensis HMD 3054T (96.0 %), Echinicola pacifica KMM 6172T (96.0 %) and Echinicola rosea JL 3085T (95.8 %). The predominant respiratory quinone was menaquinone MK-7. The major fatty acids were iso-C15 : 0, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C17 : 1ω6c and summed feature 9 (comprising iso-C17 : 1ω9c and/or 10-methyl C16 : 0). The polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid and three unidentified lipids. The DNA G+C content was 44.6 mol%. DNA-DNA relatedness between strain 001-Na2T and 'E. shivajiensis' JCM 17847T was 20.8-21.1 %. On the basis of the phenotypic features, and phylogenetic and DNA-DNA hybridization analyses presented, strain 001-Na2T (=KCTC 52495T=JCM 31786T=DSM 103729T) is the type strain of a novel species of the genus Echinicola, for which the name Echinicola sediminis sp. nov. is proposed.

Nocardioides litoris sp. nov., isolated from the Taean seashore.

A novel Gram-stain-positive, irregularly rod-shaped actinomycete, designated strain 002-2T, was isolated from sand beach sediment collected from the Taean seashore, Republic of Korea. Cells were aerobic, oxidase-negative and catalase-positive. Colonies of cells were bright yellow, circular, smooth and convex. The diagnostic diamino acid in the cell wall was ll-diaminopimelic acid. The predominant menaquinone was MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown phospholipid and two unknown amino phospholipids. The major fatty acids were iso-C16 : 0 and C17 : 1ω8c. The DNA G+C content was 68.8 mol%. 16S rRNA gene sequence analysis revealed that strain 002-2T belongs to the family Nocardioidaceae and formed a cluster with Nocardioides rubroscoriae Sco-A25T (98.1 % sequence similarity) and Nocardioides plantarum NCIMB 12834T (97.6 %). On the basis of the phenotypic and phylogenetic data, strain 002-2T (=KCTC 39838T=DSM 103718T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides litoris sp. nov. is proposed.

Characteristics of long-term changes in microbial communities from contaminated sediments along the west coast of South Korea: Ecological assessment with eDNA and physicochemical analyses.

The environmental DNA (eDNA) metabarcoding was applied to assess benthic ecological health in the west coast of South Korea by investigating a long-term microbial community change (2015-17). The ecological interaction among microorganisms, from phylum to family level, and their associations to environmental variables across the five regions were highlighted. As part of the study, the available chemistry and toxicological data in the regions during the monitoring periods were incorporated into an integrated sediment triad assessment. The bacterial communities were dominated by Proteobacteria (34.2%), Bacteroidetes (13.8%), and Firmicutes (10.8%). Proteobacteria and Bacteroidetes dominated consistently across regions and years, while Firmicutes and Cyanobacteria significantly varied by region and years (p < 0.05). The abundance of this phylum declined over time with the increasing abundance of Cyanobacteria, indicating their independent interactions to certain environmental changes. Planctomycetes and Gemmatimonadetes linked to some contaminants (ΣPAHs and Cu), implying indicator taxa. Overall, eDNA-based microbial community analysis combined with exposures of contaminants and responses of microorganisms is a promising strategy for the assessment of benthic ecological health in contaminated sediments from coastal waters.

Integrated assessment of west coast of South Korea by use of benthic bacterial community structure as determined by eDNA, concentrations of contaminants, and in vitro bioassays.

During the past few decades, contamination of sediments by persistent toxic substances (PTSs) has been observed in estuarine and coastal areas on the west coast of South Korea. The contaminants are suspected to cause toxicities in aquatic biota, but little is known about their ecological effects, particularly on benthic microbial communities. In this study, an eDNA-based assessment was applied along with classic assessments of exposure, such as chemistry and in vitro bioassays, to evaluate condition of benthic bacterial communities subjected to PTSs. Two strategies were adopted for the study. One was to conduct a comprehensive assessment in space (by comparing seawater and freshwater sites at five coastal regions) and in time (by following change over a 5-y period). Although we found that bacterial composition varied among and within years, some phyla, such as Proteobacteria (28.7%), Actinobacteria (13.1%), Firmicutes (12.7%), and Chloroflexi (12.5%) were consistently dominated across the study regions. Certain bacterial groups, such as Firmicutes and Verrucomicrobia have been linked to contamination at some sites in the study area and at specific points in time. Bacterial communities were not significantly correlated with salinity or AhR- and ER-mediated potencies, whereas concentrations of PAHs, APs, and certain metals (Cd and Hg) exhibited significant associations to the structure of bacterial communities at the phylum level. In fact, the relative abundance of microbes in the phylum Planctomycetes was significantly and negatively correlated with concentrations of PAHs and metals. Thus, the relative abundance of Planctomycetes could be used as an indicator of sedimentary contamination by PAHs and/or metals. Based on our correlation analyses, Cd and ER-mediated potencies were associated more with bacterial abundances at the class taxonomic level than were other PTSs and metals. Overall, the eDNA-based assessment was useful by augmenting more traditional measures of exposure and responses in a sediment triad approach and has potential as a more rapid screening tool.

Recovery of the benthic bacterial community in coastal abandoned saltern requires over 35 years: A comparative case study in the Yellow Sea.

Salt is an essential nutrient for humans, and salterns exist worldwide. Although the construction of salterns has stopped and typical salterns are now mostly abandoned, there has been no research on the ecological recovery of the abandoned salterns. Here, we analyzed the bacterial diversity and community structure in three pairs of abandoned salterns that have undergone 1-35 years of natural restoration and tidal flats to determine the recovery time and process. Partial 16S rRNA sequences were amplified and sequenced to investigate the biodiversity and structure of the bacterial community in sediments collected from abandoned salterns and adjacent natural tidal flats (viz., controls) in the Yellow Sea. The most abundant microorganisms across locations were found to be members of Proteobacteria, ranging from 45 to 72%, which was also a crucial taxon in the bacterial recovery process. The benthic bacterial community of the salterns showed time-dependent recovery, as demonstrated by the similarity between the salterns and controls. Indeed, dissimilarities between bacterial communities were significant for the saltern that had been abandoned for one year, according to ANOSIM (R = 1.0, p < 0.01). The genera that were determined to contribute to the dissimilarity exhibited a significant correlation with the sedimentary phosphorus concentration. The dataset generally supported that the indigenous benthic bacterial community in an altered marine environment might require a considerable time to return to a natural status. Meanwhile, a delay between the recovery of the physicochemical environment and biological component was evidenced, which seemed to influence the recovery time in a site-specific manner. Overall, the present study provided new insight and understanding of the recovery of the benthic bacterial community in abandoned salterns in terms of recovery time and the associated process.

Enhanced bioremediation of nutrient-amended, petroleum hydrocarbon-contaminated soils over a cold-climate winter: The rate and extent of hydrocarbon biodegradation and microbial response in a pilot-scale biopile subjected to natural seasonal freeze-thaw temperatures.

A pilot-scale biopile field experiment for nutrient-amended petroleum-contaminated fine-grained soils was performed over the winter at a cold-climate site. The rate and extent of hydrocarbon biodegradation and microbial responses were determined and corresponded to the on-site soil phase changes (from unfrozen to partially frozen, deeply frozen, and thawed) associated with natural seasonal freeze-thaw conditions. Treated and untreated biopiles were constructed (~3500kg each) on an open outdoor surface at a remediation facility in Saskatoon, Canada. The treated biopile received N-P-K-based nutrient and humate amendments before seasonal freezing. Real-time field monitoring indicated significant unfrozen water content in the treated and untreated biopiles throughout the freezing period, from the middle of November to early March. Unfrozen water was slightly more available in the treated biopile due to the aqueous nutrient supply. Soil CO2 production and O2 consumption in the treated biopile were generally greater than in the untreated biopile. Total removal percentages for F2 (>C10-C16), F3 (>C16-C34), and total petroleum hydrocarbons (TPH) in the treated biopile were 57, 58, and 58%, respectively, of which 26, 39, and 33% were removed during seasonal freezing and early thawing between November to early March. F3 degradation largely occurred during freezing while F2 hydrocarbons were primarily removed during thawing. Biomarker-based hydrocarbon analyses confirmed enhanced biodegradation in the treated biopile during freezing. The soil treatment increased the first-order rate constants for F2, F3, and TPH degradation by a factor of 2 to 7 compared to the untreated biopile. Shifts in bacterial community appeared in both biopiles as the biopile soils seasonally froze and thawed. Increased alkB1 gene copy numbers in the treated biopile, especially in the partially thawed phase during early thawing, suggest extended hydrocarbon biodegradation to the seasonal freeze-thaw season, due to the nutrients supplied prior to seasonal freezing.

Microbial community composition and PAHs removal potential of indigenous bacteria in oil contaminated sediment of Taean coast, Korea.

The tidal flats near Sinduri beach in Taean, Korea, have been severely contaminated by heavy crude oils due to the Korea's worst oil spill accident, say the Hebei Spirit Oil Spill, in 2007. Crude oil compounds, including polycyclic aromatic hydrocarbons (PAHs), pose significant environmental damages due to their wide distribution, persistence, high toxicity, mutagenicity, and carcinogenicity. Microbial community of Sinduri beach sediments samples was analyzed by metagenomic data with 16S rRNA gene amplicons. Three phyla (Proteobacteria, Firmicutes, and Bacteroidetes) accounted for approximately ≥93.0% of the total phyla based on metagenomic analysis. Proteobacteria was the dominant phylum in Sinduri beach sediments. Cultivable bacteria were isolated from PAH-enriched cultures, and bacterial diversity was investigated through performing culture characterization followed by molecular biology methods. Sixty-seven isolates were obtained, comprising representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, and Bacteroidetes. PAH catabolism genes, such as naphthalene dioxygenase (NDO) and aromatic ring hydroxylating dioxygenase (ARHDO), were used as genetic markers to assess biodegradation of PAHs in the cultivable bacteria. The ability to degrade PAHs was demonstrated by monitoring the removal of PAHs using a gas chromatography mass spectrometer. Overall, various PAH-degrading bacteria were widely present in Sinduri beach sediments and generally reflected the restored microbial community. Among them, Cobetia marina, Rhodococcus soli, and Pseudoalteromonas agarivorans were found to be significant in degradation of PAHs. This large collection of PAH-degrading strains represents a valuable resource for studies investigating mechanisms of PAH degradation and bioremediation in oil contaminated coastal environment, elsewhere.

Decolorizing an anthraquinone dye by Phlebia brevispora: Intra-species characterization.

Remazol brilliant blue R (RBBR) is an anthraquinone dye derived from anthracene that is decolorized by a white rot fungus, Phlebia brevispora. Interestingly, P. brevispora produces two phenomena of yellowish and pinkish colors during the degradation of RBBR. Here, we characterized the decolorization of RBBR by P. brevispora. The fungus was significantly different between the two colors via UV spectrophotometry, and the morphology of the hyphae observed in the respective color culture was also entirely different. Moreover, both of the two ligninolytic enzymes, laccase and manganese-dependent peroxidase (MnP), were remarkably stimulated in the yellowish culture at the beginning of the decolorization. It is possible that the RBBR decolorizing mechanism might be primarily related to the amount of laccase and MnP produced in the yellowish culture. Thus, the decolorized color may be rapidly estimated at initial period of incubation. In addition, GeneFishing technology revealed that two genes were differentially expressed in yellowish culture.

Heterologous expression of a new manganese-dependent peroxidase gene from Peniophora incarnata KUC8836 and its ability to remove anthracene in Saccharomyces cerevisiae.

The white rot fungus Peniophora incarnata KUC8836 has received an attention as the greatest degrader of polycyclic aromatic hydrocarbons (PAHs), which are hazardous xenobiotics and recalcitrant pollutants. To characterize the mechanisms through which MnP degrades PAHs, heterologous expression of manganese-dependent peroxidase (MnP) gene pimp1 was performed in Saccharomyces cerevisiae BY4741 via the pGEM-T Easy vector, resulting in the recombinant plasmid pESC-URA/pimp1 containing the MnP signal peptide. MnP was significantly secreted into the culture medium with galactose as an active protein with higher efficiency (3.58 U mL-1) by transformants than by the wild-type S. cerevisiae. The recombinant MnP protein was shown to have a molecular weight of 44 kDa by western blotting analysis. With regard to enhancing the bioremediation of PAHs in the environment, anthracene was effectively degraded by the MnP encoded by pimp1, with a degradation rate of 6.5% when Tween 80 was added. In addition, the MnP activity of the transformant exhibited the highest efficiency (2.49 U mL-1) during the degradation. These results show that pimp1 might be useful for biodegradation and gene expression technologies at a transcriptional level, and genetic approaches can be improved by incorporating the highly ligninolytic gene pimp1 and the fungus P. incarnata KUC8836.