Microbial Community Analysis and Effects of Fe(II)/Mn(II) Ratio on Denitrification in the Mixotrophic Biological Reactor

In this study, the denitrification performance of the mixotrophic biological reactor was investigated under varying Fe(II)/Mn(II) molar ratio conditions. Results indicate that the optimal nitrate removal ratio occurred at an Fe(II)/Mn(II) molar ratio of 9:1, pH of 7, with an HRT of 10?h. When the reactor was performing under optimal conditions, the nitrate removal reached 100.00% at a rate of 0.116?mmol·L^?1·h^?1. The proportion of oxidized Fe(II) and Mn(II) reached 99.29% and 21.88%, respectively. High-throughput sequencing results show that Pseudomonas was the dominant species in the mixotrophic biological reactor. Furthermore, the relative abundance of Pseudomonas and denitrification performance was significantly influenced by variation in the Fe(II)/Mn(II) molar ratio.     

Effect of bacterial cell size on electricity generation in a single-compartmented microbial fuel cell

A single-compartmented microbial fuel cell composed of a graphite felt anode modified with Neutral Red (NR-anode) and a porous Fe(II)-carbon cathode (FeC-cathode) were compared for electricity generation from Microbacterium sp. and Pseudomonas sp. under identical conditions. Pseudomonas sp. was more than four times the size of Microbacterium sp. based on SEM images. In cyclic voltammetry, the redox reaction between Microbacterium sp and electrode was three times the rate observed between Pseudomonas sp. and the electrode based on the Y-axis (current) variation of cyclic voltammogram. The electric power generated by Microbacterium sp. was approx 3–4 times higher than that with Pseudomonas sp. during incubation for more than 150 days in the fuel cell.     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

The distribution of active iron‐cycling bacteria in marine and freshwater sediments is decoupled from geochemical gradients

Microaerophilic, phototrophic and nitrate‐reducing Fe(II)‐oxidizers co‐exist in coastal marine and littoral freshwater sediments. However, the in situ abundance, distribution and diversity of metabolically active Fe(II)‐oxidizers remained largely unexplored. Here, we characterized the microbial community composition at the oxic‐anoxic interface of littoral freshwater (Lake Constance, Germany) and coastal marine sediments (Kalø Vig and Norsminde Fjord, Denmark) using DNA‐/RNA‐based next‐generation 16S rRNA (gene) amplicon sequencing. All three physiological groups of neutrophilic Fe(II)‐oxidizing bacteria were found to be active in marine and freshwater sediments, revealing up to 0.2% anoxygenic photoferrotrophs (e.g., Rhodopseudomonas, Rhodobacter, Chlorobium), 0.1% microaerophilic Fe(II)‐oxidizers (e.g., Mariprofundus, Hyphomonas, Gallionella) and 0.3% nitrate‐reducing Fe(II)‐oxidizers (e.g., Thiobacillus, Pseudomonas, Denitromonas, Hoeflea). Active Fe(III)‐reducing bacteria (e.g., Shewanella, Geobacter) were most abundant (up to 2.8%) in marine sediments and co‐occurred with cable bacteria (up to 4.5%). Geochemical profiles of Fe(III), Fe(II), O₂, light, nitrate and total organic carbon revealed a redox stratification of the sediments and explained 75%–85% of the vertical distribution of microbial taxa, while active Fe‐cycling bacteria were found to be decoupled from geochemical gradients. We suggest that metabolic flexibility, microniches in the sediments, or interrelationships with cable bacteria might explain the distribution patterns of active Fe‐cycling bacteria.     

Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains

The biological reduction of selenium oxyanions is capable of reducing both selenate and selenite to insoluble elemental selenium. In this process, however, bacteria inevitably require expensive chemicals such as yeast extract in almost all cases. Therefore, the reduction of selenium oxyanions with inexpensive alcohol would be more practical. A Pseudomonas sp. strain 4C‐C isolated from a sludge in a wastewater treatment facility was able to reduce selenate to selenite using ethanol as an electron donor for its anaerobic respiration, but could not reduce selenite to elemental selenium. Paracoccus denitrificans JCM‐6892, on the other hand, was observed to be able to reduce selenite to elemental selenium in the presence of ethanol, but not selenate to selenite. Therefore, a mixture containing a suspension of Pseudomonas sp. strain 4C‐C and P. denitrificans JCM‐6892 cells allowed selenate to be reduced to insoluble elemental selenium via selenite in the presence of ethanol and was also capable of reducing nitrate to nitrogen gas. Aiming at simplicity of the recovery process of insoluble elemental selenium, a polymeric gel immobilized mixture of the two bacterial strains was examined using ethanol as an electron donor. The immobilized mixture could therefore reduce not only selenate to elemental selenium, but also nitrate to nitrogen gas in a single step. The gel that immobilized the microbial mixture changed its color during the process to bright red and no red elemental selenium was left in the wastewater. This indicates that the reduced elemental selenium was completely absorbed in the gel. This simple bacterial combination would therefore be effective in the presence of ethanol to reduce selenium oxyanions in various wastewaters containing selenium and the other oxyanions.     

New application of single and mixed immobilized cells for furfural biodegradation

In this study, a new application of immobilized microbial cells for biodegradation of furfural in aqueous solution was investigated using spouted bed bioreactor. Pseudomonas sp., as a single type specie as well as activated sludge as mixed cultures were individually immobilized in 3 different bio-carrier matrices which were prepared by reinforcement of natural polysaccharides including sodium alginate, guar-gum and agar-agar with polyvinyl alcohol. The results demonstrated a complete removal (100%) of furfural from aqueous solutions using immobilized cells (IC) of Pseudomonas sp., and mixed cultures as well. Recycling of used IC for furfural removal in successive treatment cycles provided significant removal rates up to 96%. In general, results revealed that IC exhibited better performance compared to free cells in regard with the removal rate of furfural, duration of biodegradation process, as well as the ability for recycling and sustaining the high concentrations of furfural.     

On the Activities of Glucoamylase Chemically Fixed to Cyanogen Bromide-activated Cellulose

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

【目的】探究不同菌浓度和亚铁浓度条件下,Acidovorax sp. strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物。【方法】构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征。【结果】在微生物介导硝酸盐还原耦合亚铁氧化的体系中,高菌浓度促进硝酸盐还原,对亚铁氧化也有一定促进作用;高浓度亚铁在低菌浓度下氧化反应速率和程度降低,但是在高菌浓度下无明显影响;亚铁浓度越高次生矿物结晶度越高,但对硝酸盐还原具有一定抑制作用。在微生物介导亚硝酸盐还原耦合亚铁氧化的体系中,高的菌浓度和亚铁浓度都会促进亚硝酸盐还原,但亚铁氧化的次生矿物会对亚硝酸盐的微生物还原产生较强的抑制作用,次生矿物的种类和结晶度主要受亚铁浓度影响。【结论】硝酸盐还原主要是生物反硝化作用,亚硝酸盐还原包含生物反硝化和化学反硝化两部分,在硝酸盐体系中亚铁氧化与次生矿物生成是受生物和化学反硝化作用的共同影响,但亚硝酸盐体系中亚铁氧化与次生矿物生成主要是受化学反硝化作用影响。该研究可为深入理解厌氧微生物介导铁氮耦合反应机制提供基础数据和理论支撑。     

Optimization of Nitrate and Manganese Removal by Bacterium Pseudomonas sp. H117 in Mixotrophic Condition

Previous study has revealed that Pseudomonas sp. H117 could exhibit excellent performance on autotrophic and heterotrophic denitrification in polluted groundwater. However, a novel character of simultaneous denitrification and manganese removal by the bacteria remained to be further explored. In this study, we investigated optimum conditions of nitrate and Mn(II) removal by the strain H117 in mixotrophic condition. Different factors (temperature, initial pH, nitrate concentration, and Mn(II) concentration) were investigated and optimized by response surface methodology (RSM), demonstrating that the highest nitrate removal ratio (100%) in the mixotrophic condition occurred at the temperature of 30.20?°C, pH of 6.90, and Mn(II) concentration of 61.81?mg/l. Meanwhile, the optimal Mn(II) removal (73.34%) conditions were at the temperature of 29.33?°C, pH of 7.22, and nitrate concentration of 20.74?mg/l. Furthermore, microbial development pattern, cellular metabolites, and bioprecipitation were characterized by the excitation emission matrix (EEM), meteorological chromatography analysis, and scanning electron microscopy (SEM) methods, respectively. These results demonstrated that strain H117 can have good adaptability to the environment, thus exhibiting an efficient ability for bioremediation of groundwater polluted by nitrate and Mn(II).     

Reduction of Hexavalent Chromium by Immobilized Viable Cells of Arthrobacter sp. SUK 1201

Arthrobacter sp. SUK 1201, a potent isolate reported from chromite mine overburden of Orissa, India, has been evaluated for Cr(VI) reduction with immobilized whole cells. For whole-cell immobilization, Ba-alginate was found to be most effective, and the Cr(VI) reduction potential was maximum in minimal salts (MS) medium with cells immobilized in 2% alginate. Fourier transform infrared spectra of depolymerized cells has failed to detect any sign of complexation of Cr(VI) or its reduced products with the cell mass. Reduction efficiency of the beads increased with increase in cell load, but decreased with increase in Cr(VI) concentration in the medium. Glycerol was the most potent electron donor for chromate reduction, followed by glucose and peptone. Optimum pH for Cr(VI) reduction was 7.0, and the process was inhibited by metal ions such as Ni(II), Co(II), Cd(II), Zn(II), and Mn(II) but not by Cu(II) and Fe(III). Similarly, CCCP (carbonyl cyanide-m-chlorophenylhydrazone), DCC (N,N,-dicyclohexylcarbodiimide), sodium azide, and sodium fluoride were inhibitory in nature, whereas chromate reduction was unaffected in the presence of DNP (2,4-dinitrophenol). Moreover, immobilized cells of SUK 1201 remained biologically active for four consecutive cycles, accompanied with an initial increase in cell number in the beads, although a decline in chromate reduction was recorded from the second cycle onward. Immobilized cells of Arthrobacter sp. SUK 1201, therefore, could be a potential tool for long-term uses in chromium detoxification.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Oxidation of Fe(II)‐EDTA by nitrite and by two nitrate‐reducing Fe(II)‐oxidizing Acidovorax strains

The enzymatic oxidation of Fe(II) by nitrate‐reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co‐substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)‐EDTA abiotically, but the addition of the mixotrophic nitrate‐reducing Fe(II)‐oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)‐EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)‐EDTA by nitrite, resulting in the formation of Fe(III)‐EDTA and the green Fe(II)‐EDTA‐NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mm nitrate, 5 mm acetate, and approximately 10 mm Fe(II)‐EDTA resulted only in incomplete Fe(II)‐EDTA oxidation of 47–71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)‐EDTA‐NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)‐EDTA‐NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe‐EDTA species and the further reduction of the Fe(II)‐EDTA‐NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate‐reducing Fe(II)‐ and Fe(II)‐EDTA‐oxidizing cultures and demonstrates that Fe(II)‐EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)‐oxidizers.     

Alkaline iron(III) reduction by a novel alkaliphilic,halotolerant, <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from salt flat sediments of Soap Lake

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO₂. Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O₂ as alternative electron acceptors. Optimum growth was observed at 30°C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996     

Effect of Reduced Sulfur Species on Chemolithoautotrophic Pyrite Oxidation with Nitrate

We compared the response at neutral pH of some denitrifiers to different electron donors such as reduced sulfur (pyrite, S(0), and marcasite) and reduced Fe. Chemolithoautotrophic oxidation of pyrite with nitrate as electron acceptor was not possible when the pyrite was in a pure crystalline form, whereas oxidation of synthesized FeS₂ of low crystallinity and of S(0) with nitrate as electron acceptor was possible. Neither nitrite nor sulfate was formed when Fe(II)-oxidizing strain Acidovorax sp. BoFeN1 was tested. Microbial reduction of nitrate appears to be induced via S oxidation but not via Fe oxidation.     

Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^?1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^?1. Through a treatment of the immobilized cells with 0.3 mol L^?1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated–batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Autotrophic denitrification by nitrate-dependent Fe(II) oxidation in a continuous up-flow biofilter

A continuous-upflow biofilter packed with sponge iron was constructed for nitrate removal under an anaerobic atmosphere. Microbacterium sp. W5, a nitrate reducing and Fe(II) oxidizing strain, was added to the biofilter as an inoculum. The best results were achieved when NO₃ ^?-N concentration was 30 mg/L and Fe²⁺ was 800 mg/L. Nitrite in influent would inhibit nitrate removal and aqueous Fe²⁺ resulted in encrustation. Fe(II)EDTA would prevent cells from encrustation and the maximum nitrogen removal efficiency was about 90 % with Fe(II)EDTA level of 1100 mg/L. Nitrate reduction followed first-order reaction kinetics. Characteristics of biofilms were analyzed by X-ray fluorescence spectroscopy.     

Optimization of metallo-keratinase production by Pseudomonas sp. LM19 as a potential enzyme for feather waste conversion

Locally isolated bacterium Pseudomonas sp. LM19, a metallo-keratinase producer was used to hydrolyze the highly rigid keratin recalcitrant in this study. The production of crude keratinase by Pseudomonas sp. LM19 is influenced by both physical and nutritional parameters. The highest keratinase activity of 127?U/ml (2.15-fold) was observed in feather meal medium supplemented with fructose and peptone at a C/N ratio of 40. The optimum pH and temperature for keratinase production were found to be pH 8 and 30?°C, using 1% (w/v) feather as substrate. The degradation rate of the feathers was increased 2.4-fold at optimized physical and nutritional conditions. Feather degradation by Pseudomonas sp. LM19 led to the production of free amino acids such as arginine, glycine, leucine, and serine. The information on the production of keratinase by Pseudomonas sp. LM19 obtained from this study warrants further research for possible commercial application.