Novosphingobium Sp Research Articles

Conversion of estriol to estrone: A bacterial strategy for the catabolism of estriol

Natural estrogens, including estrone (E1), 17β-estradiol (E2), and estriol (E3), are potentially carcinogenic pollutants commonly found in water and soil environments. Bacterial metabolic pathway of E2 has been studied; however, the catabolic products of E3 have not been discovered thus far. In this study, Novosphingobium sp. ES2-1 was used as the target strain to investigate its catabolic pathway of E3. The metabolites of E3 were identified by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) combined with stable 13C3-labeling. Strain ES2-1 could almost completely degrade 20 mg∙L-1 of E3 within 72 h under the optimal conditions of 30°C and pH 7.0. When inoculated with strain ES2-1, E3 was initially converted to E1 and then to 4-hydroxyestrone (4-OH-E1), which was then cleaved to HIP (metabolite A6) via the 4, 5-seco pathway or cleaved to the B loop via the 9,10-seco pathway to produce metabolite with a long-chain ketone structure (metabolite B4). Although the ring-opening sequence of the above two metabolic pathways was different, the metabolism of E3 was achieved especially through continuous oxidation reactions. This study reveals that, E3 could be firstly converted to E1 and then to 4-OH-E1, and finally degraded into small molecule metabolites through two alternative pathways, thereby reducing E3 pollution in water and soil environments.

Microalgal-bacterial treatment of ice-cream wastewater to remove organic waste and harvest oil-rich biomass.

The diversity of microalgae and bacteria allows them to form beneficial consortia for efficient wastewater treatment and nutrient recovery. This study aimed to evaluate the feasibility of a new microalgal-bacterial combination in the treatment of ice cream wastewater for biomass harvest. The bacterium Novosphingobium sp. ICW1 was natively isolated from icecream wastewater and the microalga Vischeria sp. WL1 was a terrestrial oil-producing strain of Eustigmatophyceae. The ice cream wastewater was diluted 4 folds for co-cultivation, which was relatively less inhibitory for the growth of Vischeria sp. WL1. Four initial algal-bacterial combinations (v:v) of 150:0 (single algal cultivation), 150:1, 150:2, and 150:4 were assessed. During 24days of co-cultivation, algal pigmentation was dynamically changed, particularly at the algal-bacterial combination of 150:4. Algal growth (in terms of cell number) was slightly promoted during the late phase of co-cultivation at the combinations of 150:2 and 150:4, while in the former the cellular oil yield was obviously elevated. Treated by these algal-bacterial combinations, total carbon was reduced by 67.5 ~ 74.5% and chemical oxygen demand was reduced by 55.0 ~ 60.4%. Although single bacterial treatment was still effective for removing organic nutrients, the removal efficiency was obviously enhanced at the algal-bacterial combination of 150:4. In addition, the harvested oils contained 87.1 ~ 88.3% monounsaturated fatty acids. In general, this study enriches the biotechnological solutions for the sustainable treatment of organic matter-rich food wastewater.

Decrease in beneficial bacteria and increase in harmful bacteria in Gastrodia seedlings and their surrounding soil are mainly responsible for degradation of Gastrodia asexual propagation.

Asexual reproduction of Gastrodia elata Bl. f. glauca S. chow (GeB) produces degeneration with increasing number of GeB. Therefore, we analyzed the microorganisms of GeB seedlings and surrounding soil by Illumina Miseq high-throughput sequencing technology. In this study, Illumina Miseq high-throughput sequencing technology was applied to analyze the types and quantities of GeB seedlings and surrounding soil microorganisms in the first to third generations of asexual reproduction, isolated and identified the dominant strains of GeB in the first to third generations and screened the antagonistic bacteria of its pathogenic fungi, and evaluated the effects of beneficial bacteria on the production performance of seedlings planted with GeB. With an increase in the number of asexual reproductive generations, the number of pathogenic fungi and bacteria in GeB seedlings and the surrounding soil increased, and the number of beneficial fungi and bacteria decreased. Pseudomonas sp., Agrobacterium rhizomes, and Herbaspirillum hiltneri were isolated and identified in the first generation, and Trichoderma harzianum, Penicillium viridiatum, Fusarium oxysporum, and Novosphingobium sp. Were isolated and identified in the third generation. Antagonistic strains of the three pathogenic bacterial strains were screened. In conclusion, beneficial bacteria significantly improved the production performance of asexual reproductive seedlings planted with GeB. In conclusion, our findings suggested that the microorganisms of GeB seedlings and the surrounding soil change as the number of generations of GeB reproduction increases, disrupts the microecological balance of surrounding soil and endophytic microbiomes.This study provides a theoretical basis for the degradation of asexual reproduction in GeB.

Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments,and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Isolation, identification, and biological characterization of bacterial endophytes isolated from Gunnera perpensa L.

In the present study, eleven endophytic bacterial strains, Herbaspirillum sp. (GP-SGM1, GP-SGM2, GP-SGM3, and GP-SGM11), Pseudomonas sp. (GP-SGM4, GP-SGM5), Novosphingobium sp. GP-SGM6, Chryseobacterium sp. GP-SGM7, Labedella sp. GP-SGM8, Brevibacterium sp. GP-SGM9, and Pseudomonas sp. GP-SGM10, were isolated from the rhizomes of Gunnera perpensa L. The growth kinetics, assessed through maximum growth rates (μmax) and optical density (OD) values, revealed that GP-SGM7 exhibited highest μmax values of 0.33±0.01 hours (h)-1 with an OD of 4.20±0.04. In contrast, GP-SGM11 exhibited the lowest μmax of 0.12±0.05 h-1 and the smallest OD of 1.50±0.00. In addition, the endophyte crude extracts were tested for antibacterial activity against five pathogenic strains using the disk diffusion method, with GP-SGM7 crude extracts exhibiting promising antibacterial activity against Klebsiella pneumoniae and Staphylococcus aureus. Antioxidant activity was determined by DPPH (2, 2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assays. The crude extracts of GP-SGM1, GP-SGM7, GP-SGM9, and GP-SGM10 were the most effective at scavenging DPPH radicals, with GP-SGM7 also exhibiting a high FRAP value of 0.54±0.01. These findings emphasize the therapeutic potential of endophytic bacteria from G. perpensa L. in addressing skin-related issues, including bacterial infections and free radicals.

Adaptive responses and metabolic strategies of Novosphingobium sp. ES2-1–17β-estradiol analyzed through integration of genomic and proteomic approaches

Environmental 17β-estradiol (E2) can cause potential harm to ecological balance and human health. Novosphingobium sp. ES2–1 is an E2-degrading bacterium previously obtained, which converts E2 to estrone (E1) and then to 4-hydroxyestrone (4-OH-E1) followed by oxidation to form metabolites with long-chain structure during upstream degradation. Herein, we found that intracellular enzymes were the major contributors to E2 biodegradation by strain ES2–1. A total of 243 proteins were dys-expressed under E2 condition, 123 were up-regulated and 120 were down-regulated thereinto. The up-regulated members of ABC transport systems, aromatics degradation, and fatty acid degradation indicated a reinforced transfer and utilization of E2. Cytochrome P450 monooxygenase (EstP1), 2-keto-4-pentenoate hydratase, pyruvate dehydrogenase, acetyl-CoA acetyltransferase, TonB-dependent receptor were involved in E2 catabolism. During downstream degradation, the metabolites with long-chain structure were decomposed adopting β-oxidation pattern and ultimately entered the TCA cycle; 2-keto-4-pentenoic acid might be an emblematic product of such process. Furthermore, E2 converting to E1 was catalyzed by 17β-dehydrogenase probably encoded by IM701_16645 or IM701_16910; 4-OH-E1 meta-cleavage was catalyzed by a dioxygenase encoded by IM701_20340 or IM701_21000 or IM701_09625. Our study provided an in-depth insight into the adaptive responses and metabolic strategies of Novosphingobium to E2.

Biodegradation of saturate fraction of crude oil and production of signature carboxylic acids

Bacteria degrading large portion of saturated hydrocarbons are important for crude oil bioremediation. This study investigates Novosphingobium sp. S1, Gordonia amicalis S2 and Gordonia terrae S5 capability of degrading wide range of saturated hydrocarbons from Congo Bilondo crude oil and discusses the degradation pathway. A parallel analytical approach combining GC-MS and LC-HRMS enabled characterization of saturated hydrocarbons and comprehensive determination of carboxylic acid metabolites produced during biodegradation, respectively. Results showed that the three strains could efficiently degrade the n-alkanes (C10–C28) as well as methyl-substituted alkanes (C11–C26). The series of mono-, hydroxy- and dicarboxylic acids identified in this study confirmed the active biodegradation of the saturate fraction and suggest their degradation was via the bi-terminal oxidation pathway. This is the first study linking these bacterial species to bi-terminal oxidation of the saturated hydrocarbons. The study highlights the potential application of the bacterial strains in the bioremediation of crude oil contaminated sites. Additionally, while carboxylic acids is indicated as a suitable and valuable metabolic biomarker, its application is considered feasible and cost effective for rapid monitoring and evaluation of hydrocarbon biodegradation.

LuxR402 of Novosphingobium sp. HR1a regulates the correct configuration of cell envelopes.

Although there is some evidence to suggest that LuxR-solo proteins participate in inter-species or even inter-kingdom communication, most of the LuxR-solo protein functions are unknown. We have characterized the LuxR402 regulator of Novosphingobium sp. HR1a, a bacterial strain with the ability to establish high numbers in the plant rhizosphere and able to degrade a wide range of polycyclic aromatic hydrocarbons. LuxR402 controls the aggregation state of the bacterial culture; cultures of a mutant strain lacking this regulator flocculate in less than 3 h without agitation. We have demonstrated that the bacterial surface of the mutant is highly hydrophobic and that the mutant cells assimilate sugars slower than the wild-type. The flocculation mechanism has been demonstrated to be involved in the survival of the strain under unfavorable conditions; the luxR402 gene is repressed and produces flocculation in the presence of salicylate, a substrate that, although being assimilated by Novosphingobium, is toxic to cells at high concentrations. The flocculation of cultures in industrial setups has mainly been achieved through the addition of chemicals; these studies open up the possibility of controlling the flocculation by regulating the level of expression of the luxR402 gene.

Characterization of phosphate and potassium solubilization, and antifungal activity of bacteria isolated from rhizosphere of Allium ascalonicum (L.) grown in Ninh Hai district, Ninh Thuan province, Vietnam

Bacteria in the rhizosphere of red onion plants grown in Ninh Hai district, Ninh Thuan province were isolated, tested for P, K solubility and antifungal ability. Bacteria were isolated and cross-cultured on two media, NBRIP containing apatite and modified Aleksandrov containing mica. Dissolved P was measured molybdate colorimetric method and dissolved K was determined based on turbidity through present of sodium tetraphenylborate. Antifungal performance was limited to selected lines and was performed by co-culture. Bacterial identification was based on two-way sequencing for 16S rDNA sequences. The five best strains out of a total of 33 isolates were TH8, TH9, TH10, VH3 and VH5, which were able to dissolve phosphate in the range of 29.4 – 54.4 mg/L P2O5, and dissolved in potassium in the range of 13.0 – 26.4 mg/L K2O, and were resistant to A. niger with inhibition efficiency from 57% – 62.6% after 7 days of co-culture. Two strains VH5 and TH8 were also capable of antagonizing F. oxysporum with the inhibition efficiency of 60.2% and 70.7%, respectively. The identification results showed that TH9, TH10 and VH5 were homologous to Enterobacter sp., TH8 was homologous to E. soli, and VH3 was homologous to Novosphingobium sp. Enterobacter and Novosphingobium had been reported to promote plant growth in addition to salt tolerance and resistance to heavy metal pollution, so these bacterial strains could be applied to the cultivation of red onions in Ninh Thuan.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC–MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

Naturally occurring lasso peptides as algicidal agents against Microcystis aeruginosa

The expansion of harmful cyanobacterial blooms (HCBs) worldwide over the past decades has been accompanied by growing environmental and societal concerns. Numerous strategies have been developed to control HCBs, among which algicidal substances produced by bacteria are one of the most promising and eco-friendly tools. In this study, we report a novel anti-cyanobacteria bioactive product, lasso peptides (LPs), derived from a microcystin-degrading bacterium, Novosphingobium sp. ERW19. Two types of LPs (designated novosphingonodin I and II) were expressed and isolated in an Escherichia coli-based heterologous expression system. Both LPs were strongly algicidal against Microcystis aeruginosa, a dominant species of HCBs. The maximum growth inhibition rates of the crude extracts of novosphingonodin I and II against M. aeruginosa were 42.38% and 49.36%, respectively. The physiological, morphological, and transcriptomic evidences indicated that the algicidal mechanism of LPs lay in disrupting the photosynthetic system of M. aeruginosa. Both LPs inhibited the growth of M. aeruginosa by reducing the Chl a content, destroying the thylakoid membranes, decreasing the maximum quantum yields, and triggering the downregulated expression of important photosynthetic genes, which led to the failure of photosynthesis. To the best of our knowledge, this is the first report on the algicidal activity of LPs against a bloom-forming species. Moreover, we demonstrated that the in vivo production of LPs is regulated by quorum sensing (QS), thus manipulation of the QS system may be a promising strategy to achieve efficient production of LPs for HCBs control.

Degradation of long-chain alkanes through biofilm formation by bacteria isolated from oil-polluted soil

Three bacteria, Novosphingobium sp. S1, Gordonia amicalis S2 and Gordonia terrae S5, were isolated from a crude oil polluted soil and investigated for their ability to utilize long-chain alkanes (C16-C30). The strains were able to use n-hexadecane, n-octadecane and paraffins as carbon and energy source, although at different extents. Growth with alkanes, resulted in the formation of a biofilm at the alkane-water interface. Growth was shown to occur only if cells had access to the alkane-water interface suggesting that biofilm formation was required to assimilate these hydrophobic substrates. The interfacial access to alkanes is also supported by the ability of the strains to adhere to n-hexadecane, with Gordonia amicalis S2 and Gordonia terrae S5 exhibiting a much stronger adhesion than Novosphingobium sp. S1. None of the strains released biosurfactant into the culture medium which is consistent with an interfacial access to alkanes. LC-MS analyses revealed the release into the extracellular medium of 8-hydroxyhexadecanedioic acid by Novosphingobium sp. S1 and Gordonia amicalis S2, and 8-hydroxyhexadecanoic acid and 7-hydroxyhexadecanoic by Gordonia terrae S5. The production of these hydroxylated carboxylic acids substantiates the degradation of n-hexadecane by the strains although their physiological significance remains to be elucidated.

Effect of Novosphingobium sp. CuT1 inoculation on the rhizoremediation of heavy metal- and diesel-contaminated soil planted with tall fescue.

Rhizoremediation is a promising method based on the synergism between plant and rhizobacteria to remediate soil co-contaminated with heavy metals and total petroleum hydrocarbons (TPHs). A plant growth-promoting (PGP) rhizobacterium with diesel-degrading capacity and heavy metal tolerance was isolated from the rhizosphere of tall fescue (Festuca arundinacea L.), after which the effects of its inoculation on rhizoremediation performance were evaluated in heavy metal- and diesel-contaminated soil planted with tall fescue. The bacterial isolate (Novosphingobium sp. CuT1) was characterized by its indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and siderophore productivity as PGP traits. CuT1 was able to grow on 1/10 LB-agar plates containing 5mM of Cu or 5mM of Pb. To evaluate the remediation effect of heavy metal- and diesel-contaminated soil by CuT1 inoculation, the experimental conditions were prepared as follows. The soil was artificially contaminated with heavy metals (Cu and Pb) at a final concentration of 500ppm. The soil was then further contaminated with diesel at final concentrations of 0, 10,000, and 30,000ppm. Finally, all plots were planted with tall fescue, a representative hyperaccumulating plant. Compared to the rhizoremediation performance of the co-contaminated soil (Cu + Pb + diesel) without inoculation, the bioavailable Cu concentrations in the soil and the tall fescue biomass were significantly increased in CuT1 inoculation. Additionally, the root growth of tall fescue was also promoted in CuT1 inoculation. Correlation analysis showed that Cu bioavailability and bioconcentration factor were positively correlated with CuT1 inoculation. The diesel removal efficiency showed a positive correlation with CuT1 inoculation, although the diesel removal was below 30%. CuT1 inoculation was positively correlated with IAA and dehydrogenase activity in the soil. Moreover, the dry biomass of the tall fescue's roots was highly associated with CuT1 inoculation. Collectively, our findings suggest that Novosphingobium sp. CuT1 can be utilized as an applicable bioresource to enhance rhizoremediation performance in heavy metal- and TPH-contaminated soils.

The quorum sensing system of Novosphingobium sp. ERN07 regulates aggregate formation that promotes cyanobacterial growth

Microbial aggregates play key roles in cyanobacterial blooms. Being a bacterial communication mechanism, quorum sensing (QS) synchronizes gene expression in a density-dependent manner and regulates bacterial physiological behavior. However, the regulatory role of QS in the formation of cyanobacteria-associated bacterial aggregates remains poorly understood. Here, we present insight into the role of QS in regulating bacterial aggregate formation in a representative bacterial strain, Novosphingobium sp. ERN07, which was isolated from Microcystis blooms in Lake Taihu. A biosensor assay showed that ERN07 exhibits significant AHL-producing capacity. Biochemical and microscopic analysis revealed that this strain possesses the ability to form aggregated communities. Gene knockout experiments indicated that the AHL-mediated QS system positively regulates bacterial aggregation. The aggregated communities possess the ability to enhance the production of extracellular polymeric substances (EPS), alter EPS composition ratios, and affect biofilm formation. The addition of aggregated substances also has a significant growth-promoting effect on M. aeruginosa. Transcriptomic analysis revealed that the aggregated substances positively regulate photosynthetic efficiency and energy metabolism of M. aeruginosa. These findings show that QS can mediate the aggregation phenotype and associated substrate spectrum composition, contributing to a better understanding of microalgal-bacterial interactions and mechanisms of Microcystis bloom maintenance in the natural environment.

Efficient enzymatic synthesis of (S)-1-(3′-bromo-2′-methoxyphenyl)ethanol, the key building block of lusutrombopag

(S)-1-(3′-Bromo-2′-methoxyphenyl)ethanol ((S)-1b) is the key precursor for the synthesis of Lusutrombopag. The bioreduction of 1-(3′-bromo-2′-methoxyphenyl)ethanone (1a) offers an attractive method to access this important compound. Through screening the available carbonyl reductases, we obtained a carbonyl reductase from Novosphingobium aromaticivorans (CBR), which could completely convert 100 ​g/L of 1a to (S)-1b. Furthermore, a carbonyl reductase from Novosphingobium sp. Leaf2 (NoCR) was identified to completely convert 200 ​g/L of 1a to (S)-1b with excellent enantioselectivity (>99% ee) and 77% isolated yield using FDH/formate system for NADH regeneration. The Km and kcat of recombinant NoCR towards 1a were 0.66 ​mmol/L and 7.5 s-1, and the catalytic efficiency kcat/Km was 11.3 ​mmol/s.L. Meanwhile, NoCR showed high catalytic activity and stereoselectivity towards acetophenone derivatives with halogen or methoxy substitution on the benzene ring, indicating that NoCR is a valuable biocatalyst with potential practical applications.

Purification and characterization of cellulase produced by Novosphingobium sp. Cm1 and its waste hydrolysis efficiency and bio-stoning potential.

The aim of the study was to purify and characterize cellulase from a previously isolated Novosphingobium sp. strain Cm1 and to evaluate its waste hydrolysis and bio-stoning efficiency. There is a growing demand for cellulase, a multipurpose enzyme widely used in industrial applications. Here, we purified cellulase from Novosphingobium sp. Cm1 by cellulose chromatography. SDS-PAGE revealed a molecular mass of 25kDa. After 18-fold purification, the cellulase had an activity of 31.4U/mg at pH of 5 and 40°C, and it retained activity at a wide range of pH and temperatures. The presence of Fe2+ and Co2+ boosted the enzyme activity by 57% and 25% respectively. The hydrolysing capacity of the strain towards cellulosic material was assessed for two paper types and the highest activity (2.6±0.05U/ml) was found with filter paper as the sole carbon source. Alterations in the structure of the papers as a result of bacterial hydrolysis were confirmed by scanning electron microscope and Fourier-transform infrared spectroscopy. The strain was also tested for its potential in various industrial applications and exhibited pectinolytic activity (6.78±0.68U/ml), xylanolytic activity (0.22±0.14U/ml) and bio-stoning ability. The highly active purified cellulase has a broad pH and temperature range. The strain possesses waste-hydrolysing ability, pectinolytic and xylanolytic ability along with bio-stoning capacity. The efficacy and versatility of the enzyme from Novosphingobium sp. Cm1 make it an excellent candidate for diverse industrial applications.

Tap water as a natural vehicle for microorganisms shaping the human gut microbiome.

Fresh potable water is an indispensable drink which humans consume daily in substantial amounts. Nonetheless, very little is known about the composition of the microbial community inhabiting drinking water or its impact on our gut microbiota. In the current study, an exhaustive shotgun metagenomics analysis of the tap water microbiome highlighted the occurrence of a highly genetic biodiversity of the microbial communities residing in fresh water and the existence of a conserved core tap water microbiota largely represented by novel microbial species, representing microbial dark matter. Furthermore, genome reconstruction of this microbial dark matter from water samples unveiled homologous sequences present in the faecal microbiome of humans from various geographical locations. Accordingly, investigation of the faecal microbiota content of a subject that daily consumed tap water for 3 years provides proof for horizontal transmission and colonization of water bacteria in the human gut.

Production of cellulase by Novosphingobium sp. Cm1 and its potential application in lignocellulosic waste hydrolysis

Management of lignocellulosic wastes in and around the municipality area requires special consideration. Continuous deposition of these wastes to the nearby areas led to gradual deterioration of the environment. The objective of this study was to produce cellulase from the bacteria isolated from the unexplored rainforest of NE-India for lignocellulosic waste hydrolysis. Based on carboxymethyl cellulose utilization and the congo red test, Novosphingobium sp. Cm1 was found to be the most promising strain out of 114 bacterial isolates and the strain was selected for further study. The optimization of the fermentative conditions for maximum enzyme activity was carried out using one factor-at-a-time strategy and the optimum pH, temperature and incubation time was recorded as pH 5, 37 °C and 96 h respectively. The maximum β–1,4–endoglucanase activity was observed with 1.5% CMC (5.1 ± 0.05 U/mL) and 0.25% yeast extract (7.6 ± 0.72 U/mL). The bacterial waste hydrolysis ability was investigated using three wastes where vegetable waste showed maximum activity of 3.4 ± 0.48 U/mL. Bacterial interaction and waste utilization were verified using Scanning Electron Microscope and Fourier-Transform infrared spectroscopy analysis. The present study confirmed the promising ability of Novosphingobium sp. to waste hydrolysis. Further investigations may lead to new possibilities for low-cost enzyme production that will help to meet the rising cellulase demand.

Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

Glyphosate-degrading behavior of five bacterial strains isolated from stream biofilms

The present study investigates the individual degrading behavior of bacterial strains isolated from glyphosate-degrading stream biofilms. In this aim, biofilms were subjected to enrichment experiments using glyphosate or its metabolite AMPA (aminomethyl phosphonic acid) as the sole phosphorus source. Five bacterial strains were isolated and taxonomically affiliated to Ensifer sp. CNII15, Acidovorax sp. CNI26, Agrobacterium tumefaciens CNI28, Novosphingobium sp. CNI35 and Ochrobactrum pituitosum CNI52. All strains were capable of completely dissipating glyphosate after 125–400 h and AMPA after 30–120 h, except for Ensifer sp. CNII15 that was not able to dissipate glyphosate but entirely dissipated AMPA after 200 h. AMPA dissipation was overall faster than glyphosate dissipation. The five strains degraded AMPA completely since formaldehyde and/or glycine accumulation was observed. During glyphosate degradation, the strain CNI26 used the C-P lyase degradation pathway since sarcosine was quantitatively produced, and C-P lyase gene expression was enhanced 30× compared to the control treatment. However, strains CNI28, CNI35 and CNI52 accumulated both formaldehyde and glycine after glyphosate transformation suggesting that both C-P lyase and/or glyphosate oxidase degradation pathways took place. Our study shows different and complementary glyphosate degradation pathways for bacteria co-existing in stream biofilms.