Comparative genomic analysis and functional investigations for MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria in ecology.

Microcystins (MCs) significantly threaten the ecosystem and public health. Biodegradation has emerged as a promising technology for removing MCs. Many MCs-degrading bacteria have been identified, including an indigenous bacterium Sphingopyxis sp. YF1 that could degrade MC-LR and Adda completely. Herein, we gained insight into the MCs biodegradation mechanisms and evolutionary dynamics of MCs-degrading bacteria, and revealed the toxic risks of the MCs degradation products. The biochemical characteristics and genetic repertoires of strain YF1 were explored. A comparative genomic analysis was performed on strain YF1 and six other MCs-degrading bacteria to investigate their functions. The degradation products were investigated, and the toxicity of the intermediates was analyzed through rigorous theoretical calculation. Strain YF1 might be a novel species that exhibited versatile substrate utilization capabilities. Many common genes and metabolic pathways were identified, shedding light on shared functions and catabolism in the MCs-degrading bacteria. The crucial genes involved in MCs catabolism mechanisms, including mlr and paa gene clusters, were identified successfully. These functional genes might experience horizontal gene transfer events, suggesting the evolutionary dynamics of these MCs-degrading bacteria in ecology. Moreover, the degradation products for MCs and Adda were summarized, and we found most of the intermediates exhibited lower toxicity to different organisms than the parent compound. These findings systematically revealed the MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria. Consequently, this research contributed to the advancement of green biodegradation technology in aquatic ecology, which might protect human health from MCs.

Fate, abundance and ecological risks of microcystins in aquatic environment: The implication of microplastics.

Microcystins are highly toxic cyanotoxins and have been produced worldwide with the global expansion of harmful cyanobacterial blooms (HABs), posing serious threats to human health and ecosystem safety. Yet little knowledge is available on the underlying process occurring in the aquatic environment with microcystins. Microplastics as vectors for pollutants has received growing attention and are widely found co-existing with microcystins. On the one hand, microplastics could react with microcystins by adsorption, altering their environmental behavior and ecological risks. On the other hand, particular attention should be given to microplastics due to their implications on the outbreak of HABs and the generation and release of microcystins. However, limited reviews have been undertaken to link the co-existing microcystins and microplastics in natural water. This study aims to provide a comprehensive understanding on the environmental relevance of microcystins and microplastics and their potential interactions, with particular emphasis on the adsorption, transport, sources, ecotoxicity and environmental transformation of microcystins affected by microplastics. In addition, current knowledge gaps and future research directions on the microcystins and microplastics are presented. Overall, this review will provide novel insights into the ecological risk of microcystins associated with microplastics in real water environment and lay foundation for the effective management of HABs and microplastic pollution.

Biosorption of U(VI) and mechanisms by live and dead cells of Sphingopyxis sp. YF1.

Heavy metal pollution seriously threatens the environment and human health. The biosorption of heavy metals has attracted worldwide attention due to its cost-effectiveness and environmental friendliness. It is significant to develop biosorbents with excellent adsorption performance. Sphingopyxis is widely used in the removal of various organic pollutants, but its potential application in heavy metal adsorption has been largely overlooked. This study investigates the biosorption of U(VI) onto live and dead cells of a Sphingopyxis strain YF1. The effects of pH, contact time and initial ion concentration on U(VI) adsorption investigated, and kinetic and isothermal models were used to fit the adsorption results. The results show that under pH 3-6, the adsorption of U(VI) by YF1 live cells increased with the increase of the pH. Both the pseudo-first order and pseudo-second order models can satisfactorily interpret the adsorption by live and dead cells. Three isothermal adsorption models (Langmuir, Freundlich, and Sips) were used to fit the adsorption process. The adsorption of uranium by live and dead cells was best fitted by the Sips model. The maximal adsorption capacities of U(VI) by live and dead cells were 140.7 mg g-1 and 205.7 mg g-1, respectively. The mechanisms of U(VI) adsorption by Sphingopyxis sp. YF1 were revealed. Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS) show that U(VI) was deposited on the surface of the bacterial cells. Fourier-transform infrared spectroscopy (FTIR) shows that amine, hydroxyl, alkyl, amide I, amide II, phosphate, carboxylates and carboxylic acids were the major functional groups that are involved in U(VI) adsorption by live and dead cells. X-ray photoelectron spectroscopy (XPS) suggests that the main functional groups of live cells involved in adsorption were O = C-O, C-OH/C-O-C and N-C = O. This study indicates Sphingopyxis sp. YF1 is a high-efficiency U(VI)-adsorbing strain, promising to remove U(VI) from aquatic environment.

Strontium alleviated the growth inhibition and toxicity caused by cadmium in rice seedlings.

Cadmium (Cd) contamination of rice is an urgent ecological and agricultural problem. Strontium (Sr) has been shown to promote plant growth. However, the effect of Sr on rice seedlings under Cd stress is currently unclear. In this work hydroponic experiments were used to assess the impact of Sr on rice seedling growth under Cd stress. The findings demonstrated that foliar application of 0.5 mg L-1 Sr had no discernible impact on the development of rice seedlings. However, Sr significantly alleviated growth inhibition and toxicity in rice seedlings when threatened by Cd. Compared with the Cd treatment (Cd, 2.5 mg L-1), the root length, shoot height, and whole plant length of rice seedlings in the Cd + Sr treatment (Cd, 2.5 mg L-1; Sr, 0.5 mg L-1) increased by 4.96 %, 12.47 % and 9.60 %, respectively. The content of Cd in rice decreased by 23.34 % (roots) and 5.79 % (shoots). Sr lessened the degree of membrane lipid peroxidation damage (lower MDA concentration) among the seedlings of rice under Cd stress by controlling the activities of antioxidant enzymes and GSH content. By changing the expression of antioxidant enzyme-encoding genes and downregulating the heavy metal transporter gene (OsNramp5), Sr reduced accumulation and the detrimental effects of Cd on rice seedlings. Our study provides a new solution to the problem of Cd contamination in rice, which may promote the safe production of rice and benefit human health.

Biodegradation of MC-LR and its key bioactive moiety Adda by Sphingopyxis sp. YF1: Comprehensive elucidation of the mechanisms and pathways.

Microcystins (MCs) are harmful to the ecology and public health. Some bacteria can degrade MCs into Adda, but few can destroy Adda. Adda is the key bioactive moiety of MCs and mainly contributes to hepatotoxicity. We had previously isolated an indigenous novel bacterial strain named Sphingopyxis sp. YF1 that can efficiently degrade MCs and its key bioactive moiety Adda, but the mechanisms remained unknown. Here, the biodegradation mechanisms and pathways of Adda were systematically investigated using multi-omics analysis, mass spectrometry and heterologous expression. The transcriptomic and metabolomic profiles of strain YF1 during Adda degradation were revealed for the first time. Multi-omics analyses suggested that the fatty acid degradation pathway was enriched. Specifically, the expression of genes encoding aminotransferase, beta oxidation (ß-oxidation) enzymes and phenylacetic acid (PAA) degradation enzymes were significantly up-regulated during Adda degradation. These enzymes were further proven to play important roles in the biodegradation of Adda. Simultaneously, some novel potential degradation products of Adda were identified successfully, including 7­methoxy-4,6-dimethyl-8-phenyloca-2,4-dienoic acid (C17H22O3), 2-methyl-3­methoxy-4-phenylbutyric acid (C12H16O3) and phenylacetic acid (PAA, C8H8O2). In summary, the Adda was converted into PAA through aminotransferase and ß-oxidation enzymes, then the PAA was further degraded by PAA degradation enzymes, and finally to CO2 via the tricarboxylic acid cycle. This study comprehensively elucidated the novel MC-LR biodegradation mechanisms, especially the new enzymatic pathway of Adda degradation. These findings provide a new perspective on the applications of microbes in the MCs polluted environment.

Immobilization of Microbes for Biodegradation of Microcystins: A Mini Review.

Harmful cyanobacterial blooms (HCBs) frequently occur in eutrophic freshwater ecosystems worldwide. Microcystins (MCs) are considered to be the most prominent and toxic metabolites during HCBs. MCs may be harmful to human and animal health through drinking water and recreational water. Biodegradation is eco-friendly, cost-effective and one of the most effective methods to remove MCs. Many novel MC-degrading bacteria and their potential for MCs degradation have been documented. However, it is a challenge to apply the free MC-degrading bacterial cells in natural environments due to the long-term operational instability and difficult recycling. Immobilization is the process of restricting the mobility of bacteria using carriers, which has several advantages as biocatalysts compared to free bacterial cells. Biological water treatment systems with microbial immobilization technology can potentially be utilized to treat MC-polluted wastewater. In this review article, various types of supporting materials and methods for microbial immobilization and the application of bacterial immobilization technology for the treatment of MCs-contaminated water are discussed. This article may further broaden the application of microbial immobilization technology to the bioremediation of MC-polluted environments.

Optimization of Biodegradation Characteristics of Sphingopyxis sp. YF1 against Crude Microcystin-LR Using Response Surface Methodology.

Sphingopyxis sp. YF1 has proven to be efficient in biodegrading microcystin (MC)-leucine (L) and arginine (R) (MC-LR); however, the optimal environmental factors to biodegrade the toxin have not been investigated. In this study, the biodegrading characteristics of strain YF1 against MC-LR were assessed under diverse environmental factors, including temperature (20, 30 or 40 °C), pH (5, 7 or 9) and MC-LR concentration (1, 3 or 5 µg/mL). Data obtained from the single-factor experiment indicated that MC-LR biodegradation by strain YF1 was temperature-, pH- and MC-LR-concentration-dependent, and the maximal biodegradation rate occurred at 5 µg/mL/h. Proposing Box-Behnken Design in response surface methodology, the influence of the three environmental factors on the biodegradation efficiency of MC-LR using strain YF1 was determined. A 17-run experiment was generated and carried out, including five replications performed at the center point. The ANOVA analysis demonstrated that the model was significant, and the model prediction of MC-LR biodegradation was also validated with the experimental data. The quadratic statistical model was established to predict the interactive effects of the environmental factors on MC-LR biodegradation efficiency and to optimize the controlling parameters. The optimal conditions for MC-LR biodegradation were observed at 30 °C, pH 7 and 3 µg/mL MC-LR, with a biodegradation efficiency of 100% after 60 min. The determination of the optimal environmental factors will help to unveil the detailed biodegradation mechanism of MC-LR by strain YF1 and to apply it into the practice of eliminating MC-LR from the environment.

The detoxification activities and mechanisms of microcystinase towards MC-LR.

Microcystins (MCs) are the most common and toxic cyanotoxins that are hazardous to human health and ecosystems. Microcystinase is the enzyme in charge of the initial step in the biodegradation of MCs. The characterization, application conditions, and detoxification mechanisms of microcystinase from an indigenous bacterium Sphingopyxis sp. YF1 towards MC-LR were investigated in the current study. The microcystinase gene of strain YF1 was most similar to Sphingomonas sp. USTB-05 and contained a CAAX-family conversed abortive Infection (ABI) domain. The microcystinase was successful obtained and purified by overexpression in Escherichia coli. The highest degradation rate of MC-LR was 1.0 µg/mL/min under the optimal condition of 30 â„ƒ, pH 7, 20 µg/mL MC-LR, and 400 µg/mL microcystinase. The MC-degrading product was identified as linearized MC-LR, which possessed a much lower inhibitory activity against protein phosphatase 2A than MC-LR. Microcystinase interacted with MC-LR via amino acid residues involved in through the formation of conventional Hydrogen Bond, Pi-Pi T-shapes, Van der Waals force, and so on. The optimal MC-degrading condition of pure microcystinase and its detoxification mechanisms against MC-LR were revealed. The toxicity of purified linearized MC-LR was explored for the first time. These findings suggest that pure microcystinase may efficiently detoxify MCs and it is promising in the bioremediation of MC-polluted environments.

Insights into the role of extracellular DNA in heavy metal adsorption.

Extracellular polymeric substances (EPS) participate in heavy metal adsorption in the aquatic environments. Extracellular DNA (eDNA) is an essential component of EPS, but its involvement in metal binding remains ambiguous. Herein, the role of eDNA in Cd(II) and Ni(II) adsorption was described using a combination of semi-quantitative and qualitative approaches. EPS were extracted from Burkholderia sp. MBR-1 and eDNA accounted for 6.9% of the total mass of EPS. The eDNA in the extracted EPS was digested using the DNase II to prepare an eDNA-free EPS sample. Potentiometric titration unveiled that the number of total binding sites of the eDNA-free EPS was 19% lower than the untreated EPS. The Cd(II) and Ni(II) adsorption capacity of the eDNA-free EPS was lower than the untreated EPS at the pH range of 4-7. At pH 7, the results of batch adsorption experiments showed that removing eDNA from EPS resulted in declines of 12.6% and 15.7% in the adsorption capacities for Cd(II) and Ni(II), respectively. Furthermore, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy unraveled that the phosphoryl groups and purines of eDNA are responsible for Cd(II) and Ni(II) complexation. The results demonstrated that eDNA plays an essential role in heavy metal adsorption.

Bioleaching and Electrochemical Behavior of Chalcopyrite by a Mixed Culture at Low Temperature.

Low-temperature biohydrometallurgy is implicated in metal recovery in alpine mining areas, but bioleaching using microbial consortia at temperatures <10°C was scarcely discussed. To this end, a mixed culture was used for chalcopyrite bioleaching at 6°C. The mixed culture resulted in a higher copper leaching rate than the pure culture of Acidithiobacillus ferrivorans strain YL15. High-throughput sequencing technology showed that Acidithiobacillus spp. and Sulfobacillus spp. were the mixed culture's major lineages. Cyclic voltammograms, potentiodynamic polarization and electrochemical impedance spectroscopy unveiled that the mixed culture enhanced the dissolution reactions, decreased the corrosion potential and increased the corrosion current, and lowered the charge transfer resistance and passivation layer impedance of the chalcopyrite electrode compared with the pure culture. This study revealed the mechanisms via which the mixed culture promoted the chalcopyrite bioleaching.

Isolation and identification of Penicillium chrysogenum strain Y5 and its copper extraction characterization from waste printed circuit boards.

Biohydrometallurgy is generally considered as a green technology for the recycling of industrial solid waste. In this study, an indigenous fungal strain named Y5 with the ability of high-yielding organic acids was isolated and applied in bioleaching of waste printed circuit boards (PCBs). The strain Y5 was identified as Penicillium chrysogenum by morphological and molecular identification. Meanwhile, we investigated that an optimal set of culturing conditions for the fungal growth and acids secretion was 15 g/L glucose with initial pH 5.0, temperature 25°C and shaking speed 120 rpm in shaken flasks culture. Moreover, three bioleaching processes such as one-step, two-step and spent medium processes were conducted to extract copper from waste PCBs. Spent medium bioleaching showed higher copper extraction percentage and it was 47% under 5%(w/v) pulp density. Transmission electron microscope (TEM) observation combining with energy dispersive analysis of X-rays (EDAX) showed that the leached metal ions did not obviously damage the hypha cells. All above results indicated that P.chrysogenum strain Y5 has the tolerance to metal ions, suggesting its potential in recycling of metals from waste PCBs in industry.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage.

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation-resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system-type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

Recycling of metals from pretreated waste printed circuit boards effectively in stirred tank reactor by a moderately thermophilic culture.

To seek a feasible technique for processing waste printed circuit boards (PCBs), pretreatment of PCBs by table separation and further bioleached by moderate thermophiles in a stirred tank reactor were investigated. The shaking table separation, conducted after grinding and sieving of PCBs, produced two fractions: metal-rich parts (RPCBs), which is more suitable for pyrometallurgy process than untreated PCBs, and metal-poor parts (PPCBs) with only 8.83% metals was then bioleached by a mixed culture of moderate thermophiles effectively. After adaptation, the mixed culture could tolerate 80 g/L PPCBs. The bioleaching results showed that metals recovery was 85.23% Zn, 76.59% Cu and 70.16% Al in only 7 days. Trace Pb and Sn were detected in the leachate because of precipitating. The microorganism community structure was analyzed by amplified ribosomal DNA restriction analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus. Furthermore, uncultured Thermoplasmatales archaeon was also detected in the leaching system. It was also shown that moderate thermophiles revealed best bioleaching ability when compared with mesophiles and the mixture of mesophiles and moderate thermophiles. Finally, we designed a two-stage process model according to the present study to achieve semi-industrial waste PCBs recycling and economic feasibility analysis indicated that the process was profitable.

Expression of Critical Sulfur- and Iron-Oxidation Genes and the Community Dynamics During Bioleaching of Chalcopyrite Concentrate by Moderate Thermophiles.

Sulfate adenylyltransferase gene and 4Fe-4S ferredoxin gene are the key genes related to sulfur and iron oxidations during bioleaching system, respectively. In order to better understand the bioleaching and microorganism synergistic mechanism in chalcopyrite bioleaching by mixed culture of moderate thermophiles, expressions of the two energy metabolism genes and community dynamics of free and attached microorganisms were investigated. Specific primers were designed for real-time quantitative PCR to study the expression of these genes. Real-time PCR results showed that sulfate adenylyltransferase gene was more highly expressed in Sulfobacillus thermosulfidooxidans than that in Acidithiobacillus caldus, and expression of 4Fe-4S ferredoxin gene was higher in Ferroplasma thermophilum than that in S. thermosulfidooxidans and Leptospirillum ferriphilum. The results indicated that in the bioleaching system of chalcopyrite concentrate, sulfur and iron oxidations were mainly performed by S. thermosulfidooxidans and F. thermophilum, respectively. The community dynamics results revealed that S. thermosulfidooxidans took up the largest proportion during the whole period, followed by F. thermophilum, A. caldus, and L. ferriphilum. The CCA analysis showed that 4Fe-4S ferredoxin gene expression was mainly affected (positively correlated) by high pH and elevated concentration of ferrous ion, while no factor was observed to prominently influence the expression of sulfate adenylyltransferase gene.

Metallosphaera tengchongensis sp. nov., an acidothermophilic archaeon isolated from a hot spring.

Two novel acidothermophilic archaea, strains Ric-A(T) and Ric-F, were isolated from muddy water samples of a sulfuric hot spring located in Tengchong County, Yunnan Province, PR China. The strains were aerobic and facultatively chemolithoautotrophic. Both strains could oxidize S(0) and K2S4O6 for autotrophic growth, and could use organic materials for heterotrophic growth. Growth was observed at 55-75 °C and pH 1.5-6.5. The strains could oxidize metal sulfide ores, showing their potential in bioleaching. The DNA G+C contents of strains Ric-A(T) and Ric-F were 41.8 and 41.6 mol%, respectively. Analysis of 16S rRNA gene sequences showed that the two strains shared 99.8 % sequence similarity to each other, but <97 % to other known species of the genus Metallosphaera. DNA-DNA hybridization indicated that the isolates were different strains of a novel species of the genus Metallosphaera. Strains Ric-A(T) and Ric-F also shared a number of physiological and biochemical characteristics that distinguished them from recognized species of the genus Metallosphaera. On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with their closest relatives, it was concluded that strains Ric-A(T) and Ric-F represent a novel species of the genus Metallosphaera, for which the name Metallosphaera tengchongensis sp. nov. is proposed. The type strain is Ric-A(T) ( = NBRC 109472(T) = CGMCC 1.12287(T)).

[Isolation and identification of Sulfobacillus sp. strains and their application in pyrite bioleaching].

OBJECTIVE: The work aimed to isolate and culture the acidophilic and moderately thermophilic microorganisms for leaching the sulfide ore. METHODS: We enriched and incubated iron- or sulfur-oxidizing strains from muddy water of acuric hot spring utilizing ferrous irons or elemental sulfur as substrates. Then, we identified the strains by their morphological, physiological, biochemical properties and phylogenetic positions, and estimated their bioleaching potential according to their oxidation rate of pyrite. RESULTS: Two acidophilic, aerobic and facultative heterotrophic bacterial strains, Costa C and Costa E, were isolated from the samples of sulfuric hot springs of Costa Rica. Cells of the two strains were gram-positive, spore-forming and rod-shaped [(0.4 - 0.6) microm x (2.5 - 4.0) microm and (0.4 - 0.7) microm x (2.4 - 4.9) microm, respectively]. Strain Costa C grew at a temperature range of 30 degrees C - 55 degrees C and a pH range of 1.2 - 5.0, optimally at 50 degrees C and 2.8. Strain Costa E grew at a temperature range of 30 degrees C - 55 degrees C and at a pH range of 1.4 - 5.0, optimally at 40 degrees C and 2.8. Two strains could autotrophically grow on inorganic substrates such as ferrous irons, element sulfur and K2 S4 O6 and also could utilize organic substrates like yeast extract for heterotrophic growth. Phylogenetic analysis based on 16S rRNA gene sequences alignment demonstrated that the highest similarity between strain Costa C, Costa E and other species of the genus Sulfobacillus was above 99%. CONCLUSION: Based on morphological, physiological and biochemical analysis, Costa C and Costa E can be affiliated to the genus Sulfobacillus, for which the names Sulfobacillus sp. strain Costa C and Sulfobacillus sp. strain Costa E were proposed. Both strains could oxidize pyrite, and the oxidation rates arrived 63.0 mg/L x d and 56.8 mg/L x d, respectively.