Impact of Anthropogenic Activities on Microbial Community Structure in Riverbed Sediments of East Kazakhstan.

Heavy metal (HMe) pollution in regions with mining and metallurgy activities is known to be a serious environmental problem worldwide. Hydrological processes contribute to the dissemination of HMes (drainage, precipitation, flow rate). The aim of the present study is to investigate the microbial community structure in ten river sediments sampled in different regions of East Kazakhstan, which are contaminated with HMes. The overall degree of sediment contamination with HMes (Cr, Cu, Zn, Pb, and Cd) was assessed using the pollution index Zc, which ranged from 0.43 to 21.6, with the highest in Ridder City (Zc = 21.6) and Ust-Kamenogorsk City, 0.8 km below the dam of the hydroelectric power station (Zc = 19.6). The tested samples considerably differed in organic matter, total carbon, nitrogen, and phosphorus content, as well as in the abundance of HMe-related functional gene families and antibiotic resistance genes. Metagenomic analysis of benthic microorganisms showed the prevalence of Proteobacteria (88.84-97.61%) and Actinobacteria (1.21-5.98%) at the phylum level in all samples. At the class level, Actinobacteria (21.68-57.48%), Betaproteobacteria (19.38-41.17%), and Alphaproteobacteria (10.0-39.78%) were the most common among the classified reads. To the best of our knowledge, this is the first study on the metagenomic characteristics of benthic microbial communities exposed to chronic HMe pressure in different regions of East Kazakhstan.

Current Trends in Bioaugmentation Tools for Bioremediation: A Critical Review of Advances and Knowledge Gaps.

Bioaugmentation is widely used in soil bioremediation, wastewater treatment, and air biofiltration. The addition of microbial biomass to contaminated areas can considerably improve their biodegradation performance. Nevertheless, analyses of large data sets on the topic available in literature do not provide a comprehensive view of the mechanisms responsible for inoculum-assisted stimulation. On the one hand, there is no universal mechanism of bioaugmentation for a broad spectrum of environmental conditions, contaminants, and technology operation concepts. On the other hand, further analyses of bioaugmentation outcomes under laboratory conditions and in the field will strengthen the theoretical basis for a better prediction of bioremediation processes under certain conditions. This review focuses on the following aspects: (i) choosing the source of microorganisms and the isolation procedure; (ii) preparation of the inoculum, e.g., cultivation of single strains or consortia, adaptation; (iii) application of immobilised cells; (iv) application schemes for soil, water bodies, bioreactors, and hydroponics; and (v) microbial succession and biodiversity. Reviews of recent scientific papers dating mostly from 2022-2023, as well as our own long-term studies, are provided here.

Batch-mode stimulation of hydrocarbons biodegradation in freshwater sediments from historically contaminated Aluksne lake.

Historical contamination of freshwater lakes with hydrocarbons (HC) due to anthropogenic activities represents a serious problem worldwide. This study was focused on hydrocarbons-contaminated sediments sampled in Lake Aluksne of glacial origin in Northeast Latvia. The batch experiments were aimed at evaluating the effect of bio-stimulation and bioaugmentation on the biodegradation of hydrocarbons in lake sediments (LS), as well as changes in microbial community structure and metabolic activity. The sediments were sampled from two points of the lake, 4-5 m and 8 m depth, respectively. These samples slightly differed by colour, count of diatoms, microbial respiration intensity and colour intensity of 2,6- dichlorophenolindophenol. Nevertheless, the trend in biodegradation activity was similar for both LS samples. The concentration of HC in LS during the 32-day incubation decreased in average from 465 mg/kg to 165 mg/kg and 117.5 mg/kg in the LS amended with nutrients and nutrients+microbial community, respectively. Different treatment types of LS resulted in differences in microbial respiration and HC-degrading activity. The Shotgun sequencing has revealed the main phyla present in the intact LS being Proteobacteria (48.8%), Actinobacteria (24.4%), Firmicutes (10.4%) and Bacteroidetes (5.0%). Incubation of LS for 32 days resulted in increasing abundance of Proteobacteria from 48.8% in the raw LS to 58-62%, mainly due to the increase of Betaproteobacteria. The functional annotation of gene families revealed that the most abundant gene families were associated with ATP binding, metal ion, magnesium ion, sulfur cluster, zinc ion binding, DNA binding and other essential components for cell functioning. The Shannon biodiversity index of culturable microorganisms in EcoPlates™ ranged from 2.28 to 2.85. The data obtained in this study indicated that the suggested approach is a potent remediation technology for further ex situ scaling up.

Ca2Fe2O5 powder antifungal activity to the Candida utilis culture upon its growth.

This study reports the impact of Ca2Fe2O5 porous powder on the yeast Candida utilis-as a fungal model-at different phases of growth, i.e., early exponential (6 h), mid-log (11 h), and stationary (17 h) phases. Ca2Fe2O5 inhibited the cell growth in a time-dependent manner. After 120 min incubation, the fungicidal activity of porous powder was observed, i.e., log reduction of 2.81 and 2.58 for 11 and 17 h cultures, respectively, reaching the maximum of 4 log reduction after 7 days. Nevertheless, the 6 h culture of C. utilis showed enhanced resistance to Ca2Fe2O5 with a ≤ 0.4 log reduction during the 7 days exposure. Our results not only showed that Ca2Fe2O5 has the potential to effectively eliminate the C. utilis cell growth but also indicated the importance of the yeast culture physiological state for resistance to Ca2Fe2O5. To the best of our knowledge, this is the first study that evaluated the fungicidal activity of Ca2Fe2O5 porous powder on C. utilis and the impact of the C. utilis phase of growth on the cell susceptibility.

Stimulation of sewage sludge treatment by carbon sources and bioaugmentation with a sludge-derived microbial consortium.

Recently, sewage sludge (SS) disposal has become one of the greatest global challenges. In this study, we aimed to evaluate the effect of faba bean straw (Straw-B), wheat straw (Straw-W), and wood-chip pellets (WCP) amended to SS, as well as bioaugmentation (BA), on the physicochemical characteristics and structure of the microbial community of the treated SS. Sixteen days of incubation of SS-containing mixtures revealed the highest efficiency of Straw-W(BA) in terms of SS stabilisation, i.e., the highest and most stable respiration intensity, the lowest ammonia emission, and the highest stimulation effect on the cress seedling growth. Shotgun sequencing data analysis showed that Proteobacteria dominated in the raw SS with 60.17% reads, which consisted of 16.40%, 29.18%, and 12.33% of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, respectively. All treated samples were characterised by an increased abundance of Firmicutes (32.70-53.84%). A remarkable increase in virus abundance (0.34% reads) was detected in the treated SS, which was incubated without C amendment and bioaugmentation. The addition of C sources to the SS changed some physicochemical characteristics of the mixture. All of these findings provide novel insights toward a mechanistic understanding of the fate of the human sewage microbiome in wastewater and other environments.

Selective enrichment of heterotrophic nitrifiers Alcaligenaceae and Alcanivorax spp. from industrial wastewaters.

Removal of nitrogen from wastewaters (WW) represents a global problem. The low nitrification rate during WW treatment is often caused by ecotoxicity. This problem is attributed mostly to the industrial WW. Our study was focused on the testing of industrial WW and activated sludge (AS) with the aim to reveal the abundance of nitrifiers and increase their biomass, thus, providing the additional step, i.e., bioaugmentation, within the technological process of WW treatment. Plating of AS on the selective solidified media designated for the 1st and 2nd nitrification stages, resulted in the shift in bacterial community structure with dominated Alcaligenaceae and Alcanivorax for the 1st stage, and Alcanivorax-for the 2nd stage of nitrification, respectively. Incubation of AS in the presence of real WW and selective nitrification broth resulted in a considerable increase (one or two magnitudes in the presence of the 1st and 2nd stage nitrification broth, respectively) of culturable nitrifiers after 5 days incubation under aerated conditions. The obtained data provide with evidence about a possibility to strengthen the role of heterotrophic nitrifiers in the treatment of industrial WW, where toxicity obstacles inhibited nitrification under conventional conditions.

Modeling the mobility of glyphosate from two contrasting agricultural soils in laboratory column experiments.

Glyphosate (GLP) currently is one of the most widely used herbicides worldwide. The persistence of GLP and its major metabolite, aminomethylphosphonic acid (AMPA) in the environment has been described by other authors. This study was aimed at comparing the GLP and AMPA behavior in sandy and loamy sand soils after spiking with enhanced (445 µg g-1) concentrations of GLP in herbicide KLINIK® (Nufarm, Austria) and bioaugmentation followed by 40 days weathering and a consistent three-stage leaching in a laboratory column experiment. Soil samples were obtained from mineral topsoil (0-10 cm) within former agricultural lands where soil parent material was formed by glacigenic deposits. The total amount of GLP and AMPA collected during three leaching stages was significantly (p<.05) higher from columns with sandy soil, compared to loamy sand soil. Bioaugmentation resulted in considerably lower concentrations of AMPA in leachates, especially in the sets with sandy soil (p=.01). Leachates were tested using FTIR spectroscopy and Daphnia magna. Statistical analysis of the changes in Ntot, Ctot, K+, Mg2+, Al3+, Ca2+, Mn2+ and Fe3+ concentrations in soils after the leaching experiment revealed that the loamy sand soil was likely to be more sensitive to the addition of GLP and bioaugmentation than sandy soil.

Metabolic response of bacteria to elevated concentrations of glyphosate-based herbicide.

Glyphosate-based herbicides (GBHs) are the most widespread commonly used broad-spectrum herbicides that contaminate soils and waters, are toxic to bacteria, plants and animals, and have been classified as 'probably carcinogenic to humans' by the International Agency for Research on Cancer in 2015. Particular soil bacteria and fungi can degrade GBHs, hence, search for new GBH-degrading strains or microbial consortia, effective under specific growth conditions and local environment, seems to be a promising solution for bio-remediation of glyphosate-contaminated environment. Consequently, there is a need for rapid and informative methods to evaluate the GBH-induced changes of the metabolic pathways in cells, that may serve as indicators of GBH-degrading potential. Three new GBH-degrading bacterial strains, Pseudomonas sp., Actinobacteria and Serratia sp. were isolated from sludge of municipal waste water treatment plant (Daugavgriva, Riga, Latvia), agricultural soil and plant tissue, respectively. This study examined the response of these isolates to elevated concentrations of glyphosate (GLP) (100 and 500 mg/L) in GBH Klinik® 360 SL. The GBH-induced shift of metabolic activity in cells of Pseudomonas sp. was shown by tests on EcoPlates™. Fourier transform infrared (FTIR) spectroscopy analyses were used to evaluate the metabolomic response of bacteria to elevated concentrations of GBH in the growth environment. The spectra of Pseudomonas sp. and Serratia sp., incubated with and without GBH, were similar, thus indicating their GBH-resistance. The absorption at 1736 cm-1, assigned to ester carbonyl stretch vibrations, was detected in spectra of all three bacteria. The highest ester content was detected in Actinobacteria grown in medium with 1.0% molasses and 100 or 500 mg/L GLP in GBH Klinik®. An increase of cellular amounts of esters, either those of phospholipids or poly-ß-hydroxybutyrates, indicates degradation of GLP. Therefore, monitoring the ester carbonyl stretch vibration band in FTIR spectra of bacterial biomass may speed up the search GBH-degrading strains. Microbiological tests and cell metabolic response studies by FTIR spectroscopy showed that the three new isolates of Pseudomonas sp., Actinobacteria and Serratia sp. were resistant to elevated concentrations of GBH Klinik® in growth environment and exhibited the potential for GBH degradation.

The effects of ibuprofen on activated sludge: Shift in bacterial community structure and resistance to ciprofloxacin.

Ibuprofen (IBP) is ranked at the 4th place among 57 pharmaceutical compounds according to the number of citations in prioritization documents. The response of microbial community of activated sludge to IBP was studied at the concentrations of 50-5000mg/L. Batch incubation was performed in an OxiTop® device for 21days. The reduction of biological oxygen demand depended on the IBP concentration and varied in the range from 321 to 107mg O2/L. Massive DNA sequencing analysis of the activated sludge revealed that Proteobacteria became more dominant when grown in the presence of IBP. Microbial diversity was reduced in the presence of 500-1000mg/L IBP, but increased again in the presence of 5000mg/L IBP, despite the domination of Enterobacteriales (48.1%) in this sample. Incubation of activated sludge in the presence of 1000mg/L IBP led to an increased occurrence of ciprofloxacin-resistant bacteria. The use of Eosin Methylene Blue Agar for disc diffusion assay was shown to be more appropriate in order to reveal the changes in antibiotic resistance. The predominance of Enterobacteriales in the activated sludge is suggested as one of the possible explanations of the enhanced resistance to ciprofloxacin.

Removal of pharmaceuticals from municipal wastewaters at laboratory scale by treatment with activated sludge and biostimulation.

Municipal wastewater containing 21 pharmaceutical compounds, as well as activated sludge obtained from the aeration tank of the same wastewater treatment plant were used in lab-scale biodegradation experiments. The concentrations of pharmaceutical compounds were determined by high-performance liquid chromatography coupled to Orbitrap high-resolution mass spectrometry and ranged from 13.2ng/L to 51.8µg/L. Activated sludge was characterized in the terms of phylogenetic and catabolic diversity of microbial community, as well as its morphology. Proteobacteria (24.0%) represented the most abundant phylum, followed by Bacteroidetes (19.8%) and Firmicutes (13.2%). Bioaugmentation of wastewater with activated sludge stimulated the biodegradation process for 14 compounds. The concentration of carbamazepine in non-amended and bioaugmented WW decreased during the first 17h up to 30% and 70%, respectively. Diclofenac and ibuprofen demonstrated comparatively slow removal. The stimulating effect of the added nutrients was observed for the degradation of almost all pharmaceuticals detected in WW. The most pronounced effect of nutrients was found for erythromycin. The results were compared with those obtained for the full-scale WW treatment process.

The effects of woodchip- and straw-derived biochars on the persistence of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in soils.

Sorption and degradation are the primary processes controlling the efficacy and runoff contamination risk of agrochemicals. This study assessed the influence of two biochars, made from woodchips and straw at a pyrolysis temperature of 725°C and applied to a loamy sand and a sandy soil in the concentration of 5.3 g 100 g(-1) sandy soil and 4.1 g 100 g(-1) loamy sand soil, or 53 t ha(-1) for both soil types, on degradation of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Soils were spiked with 50 mg MCPA kg(-1) soil. In the sandy soil, significantly more MCPA remained after 100 days if amended with straw-derived biochar in comparison to wood-derived biochar. Both biochars types significantly increased urease activity (p<0.05) after 37 days in the loamy sand soil, but these differences disappeared after 100 days. A root and shoot elongation test demonstrated that the soils containing straw-derived biochar and spiked with MCPA, showed the highest phytotoxicity. Both biochars were found to retard MCPA degradation in loamy sand and sandy soils. This effect could not be explained only by sorption processes due to comparatively low developed micro/mesoporous structure of both biochars shown by BET surface analysis. However, an enhanced MCPA persistence and soil toxicity in sandy soil amended with straw biochar was observed and further studies are needed to reveal the responsible mechanisms.

The role of nutrients in the biodegradation of 2,4,6-trinitrotoluene in liquid and soil.

The widely used explosive 2,4,6-trinitrotoluene (TNT) has residues that are potentially explosive, toxic, and mutagenic. TNT and other explosives can be degraded by microorganisms; however, biostimulation is needed for process efficiency. To investigate the effectiveness of using biostimulation to degrade TNT, we added varying concentrations of a nutrient amendment consisting of inorganic salts, plant extracts, and molasses to soil and liquid media. For the inoculum we used a consortium of bacteria AM 06 that had exhibited the ability to degrade TNT and which had been previously isolated from explosives-contaminated soils. Phylogenetically, the clones clustered into seven different genera: Klebsiella, Raoultella, Serratia, Stenotrophomonas, Pseudoxanthomonas, Achromobacter and Pseudomonas. The addition of AM 06 consortium to a liquid environment along with 100% nutrient amendment decreased the amount of TNT (and its degradation products) by up to 90% after 14 days incubation. At the total amount of TNT was less than 100 mg/l, the concentration of TNT did not influence the amount of sugar consumed by the bacteria consortium. In soil media, the TNT degradation process was dependent on the concentration of nutrient amendment added. At higher initial concentrations of TNT (500 mg/kg), bioaugmentation (i.e., addition of bacteria inoculum) had a demonstrated effect, especially when nutrient concentrations of 50% and 100% were added to the soil. Findings of this study could further the understanding of the TNT biodegradation processes in water and soil and provide for optimization of the technological conditions for bioremediation.

Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds.

Previous studies showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific consortium of bacteria upon their growth. For further development of the approach for contaminated soil remediation it was necessary to evaluate the qualitative and/or quantitative composition of different origin CLE and their relevance on the growth of explosives-degrading bacteria. Six CLE (different by species, cultivars and harvesting time) were tested and used as additives to the growth medium. It was shown that nitro aromatic compounds can be identified in the FT-IR absorption spectra by the characteristic band at 1,527 cm(-1), and in CLE by the characteristic band at 1,602 cm(-1). The intensity of the CLE band at 1,602 cm(-1) correlated with the concentration of total nitrogen (R2=0.87) and decreased upon the growth of bacteria. The content of nitrogen in CLE differed (0.22-1.00 vol.%) and significantly influenced the content of total carbohydrates (9.50-16.00% DW) and lipids [3.90-9.90% dry weight (DW)] accumulated in bacterial cells while the content of proteins was similar in all samples. Though this study showed quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media, and proved the potential of this additive for remediation of contaminated soil. It was shown that analysis of CLE and monitoring of the conversion of nitro aromatic compounds can be investigated by FT-IR spectroscopy as well as by conventional chemical methods.

Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms.

Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. A search for optimal growth conditions for soil bacteria is of a great importance in order to isolate various xenobiotic degraders. Bacteria consortium A43 was isolated from soils contaminated with explosives. In the presence of carbohydrate and plant extract, an addition of TNT to the solidified minimal medium stimulated the growth of the tested bacteria, as compared to other bacteria consortium isolated from the same soils. Reducing sugars as carbohydrates, and cabbage leaf extract as a plant extract were used in these experiments. Cultivation of the A43 in liquid medium of the same content showed that addition of cabbage leaf extract alone to medium is much more efficient for TNT degradation by growing biomass as compared to addition of carbohydrate alone.