Mixed Microorganisms Research Articles

Observation of 2,3-butanediol biosynthesis in Lys regulator mutated Klebsiella pneumoniae at gene transcription level

Microorganisms that produce 2,3-butanediol (2,3-BDO) are mostly mixed acid fermentation microorganisms, and they synthesize 2,3-BDO in order to suppress medium acidification. The 2,3-BDO operon (budBAC) is activated by the LysR regulator protein derived from the budR. In this study, the effect of the budR on 2,3-BDO-biosynthesis was observed at gene transcription level. The Klebsiella pneumoniae strains (wabG-deleted strain (SGSB100), budR over-expressed strain (SGSB101), and the budR-deleted strain (SGSB102)) were constructed. The resulting strains were cultivated in unified conditions. Samples were obtained at the lag-, log-, and stationary-phase of cell growth, and gene transcription levels of the budR, 2,3-BDO-biosynthesis-related (budB, budA, and budC), and acid-biosynthesis-related (ldhA and ack) genes were observed. During the lag-phase of cell growth in SGSB101, the budR transcription level increased approximately 8-fold, and 2,3-BDO production increased approximately 2-fold, when compared to SGSB100. Also in SGSB101 the transcription level of the acid-biosynthesis-related genes (ldhA and ack) increased approximately up to 11-fold during the lag-phase of cell growth compared to SGSB100. On contrast, in SGSB102 budR transcription was not detected, and the transcription level of the acid-biosynthesis-related genes (ldhA and ack) decreased approximately 70-fold during the lag-phase of cell growth compared to SGSB100. This is by far the first observation of 2,3-BDO regulation mechanism at gene transcription level in K. pneumoniae, and therefore may be useful for understanding and improving 2,3-BDO biosynthesis metabolic network.

Performance of straw-fed microbial fuel cells with mixed rumen microorganisms by using different catholytes

Rumen microbial fuel cells (RMFCs) are a promising technology for sustainable production of alternative energy and fiber waste treatment. However, the influence of catholytes with different oxidizing powers on anode metabolites and electrical properties of straw-fed MFCs will be mainly evaluated in this study. Furthermore, the exploration of nonlinear features in RMFCs would be another subject of debate. Results show that voltage generation correlates positively to anode total volatile fatty acids, acetic acid, and butyric acid concentrations, along with the ratio of acetic acid to propionic acid, which would favor the production of reducing equivalents. In addition, the finding of an attractor indicates that RMFCs possessing a chaotic characteristic is a nonlinear system and could be used to strengthen the metabolism of microorganisms. These observations will provide information for improvement in RMFC fed native plant fiber substrate.

Effects of mutation on a new strain Leptospirillum ferriphilum YXW and bioleaching of gold ore

Leptospirillum ferriphilum YXW was isolated through serial dilution from mixed microorganisms enriched in AMD from Dexing copper mine in Jiangxi Province, China. It was mutated by ultrasonic, UV and microwave to collect more efficient strain for bioleaching of gold ore. Physiological and biochemical characteristics indicate that strain YXW is a strict chemoautotrophic microorganism, and the optimal condition for its growth is temperature of 40 °C and pH 1.5. After mutation by ultrasonic, UV and microwave, the density of bacterial cells reached 9×109, 8.4×109 and 4.3×108 mL−1, increased by 291%, 265% and 87%, respectively, compared with the original culture. The bacterial total protein activity was improved by microwave and UV mutations, but was reduced by ultrasonic. Mutations had effects on bioleaching of gold ore in sequence of microwave > UV > ultrasonic. During gold ore bioleaching, the bacterial mutant after mutation by microwave had the best effect on the extraction rates of arsenic and iron, which were 19.6% and 17.7% higher than that of the original strain after bioleaching for 10 d, respectively. The results suggested that the effects of mutation on bioleaching of gold ore may not be mainly due to increase of bacterial cells density, but may be mainly attributed to the improvement of bacterial total protein activity.

Settling and dewatering characteristics of mixed microorganisms according to changes in the SRT

ABSTRACT This study was carried out to evaluate the settleability and dewaterability of mixed microorganisms according to the changes in sludge retention time (SRT) after mixing granulated methane-oxidizing bacteria and activated sludge (AS). The results of increasing SRT of microorganisms from 15 to 20 days showed that the settling rate of microorganisms increased, while specific resistance to filtration decreased. From the results of analyzing extracellular polymeric substances (EPS), no significant change was found in the bound EPS, but total soluble EPS showed a tendency to decrease as the SRT increased from 15 to 20 days. The average particle size of the mixed microorganisms was two to three times larger than that of the general AS, and as the SRT increased, the average particle size also increased, indicating that there is a close correlation between SRT and granule formation. The results of analyzing microbial community in SRT 15 and 20 days showed the dominance of Proteobacteria at long SRT.

Optimization and kinetics studies on biodegradation of atrazine using mixed microorganisms

In this work, degradation of atrazine was carried out in batch reactors using mixed microorganisms obtained from pharmaceutical wastewater sludge. The effects of process parameters like pH, temperature, inoculum concentration, and agitation speed on atrazine degradation were studied and optimized using response surface methodology (RSM). The optimum condition for the maximum degradation of atrazine was pH – 6.7, temperature – 29.3°C, inoculum concentration – 5%, and agitation speed – 137rpm. At these conditions, the effect of atrazine concentration was studied. From the results, it was found that increase in atrazine concentration decreases the degradation efficiency. The maximum atrazine degradation was found to be 94.4%. Various cell growth models and substrate inhibition models were used to describe the atrazine degradation kinetics. From the results, it was found that Haldane model fits the data well with R2 value of 0.9001.

Application of Statistical Tool for the Optimization of Biofiltration of Toluene Using Corn Stacks as Packing Material

In this work, statistical design was applied for the optimization of process parameters for the biofiltration of toluene using mixed microorganisms obtained from pharmaceuticals wastewater sludge in a packed bed biofilter. The effects of process parameters such as gas flow rate, initial toluene concentration, height of the packing material, and temperature on removal efficiency (RE) of toluene was studied and optimized using response surface methodology. Using Box–Behnken Design, 30 experiments were carried out for the four test variables. From the results, the optimum condition for maximum RE was found to be: gas flow rate—0.01 m3 h−1, initial toluene concentration—0.25 g m−3, packing height—0.75 m, and temperature—31 °C. At these optimized conditions, the removal efficiency of toluene was found to be 95 %. A high R 2 value of greater than 0.9 indicates the fitness of the model to predict the experimental data. Biokinetic studies led to a good fit between experimental data and mathematical equations, yielding a calculated maximum toluene degradation rate of 140.42 g m−3 h−1 with a half-velocity constant, K S, of 0.076 g m−3.

Enhanced remediation of black liquor by activated sludge bioaugmented with a novel exogenous microorganism culture

Black liquor (BL) is a notoriously difficult wastewater to treat due to the economic and efficiency limitations of physiochemical methods and intrinsic difficulties with bioremediation strategies caused by the high pH (10-13) and lignin content. This study investigated the feasibility of a novel bioaugmentation strategy for BL treatment, which uses a mixed microorganism culture of lignocellulose-degrading microorganisms isolated from degraded bamboo slips. Black liquor treatment was assessed in terms of chemical oxygen demand (COD) and color removal with a sequencing batch reactor organic loading rate of 9kg COD/L·day under highly alkaline conditions (pH 10). Results revealed that bioaugmented activated sludge treatment of BL with special mixed microorganisms significantly enhanced the removal efficiency of COD, color, and lignin from the wastewater up to 64.8, 50.5, and 53.2%, respectively. Gel permeation chromatography profiles showed that the bioaugmentation system could successfully degrade high molecular lignin fragments in black liquor. This work confirms bioaugmentation as a feasible alternative strategy for enhanced biological treatment of wastewater with high lignin content and high organic load rate under strongly alkaline conditions.

Pilot scale feasibility study for in-situ chemical oxidation using H2O2 solution conjugated with biodegradation to remediate a diesel contaminated site

A pilot scale test for a process combining in-situ chemical oxidation using H2O2 solution with biodegradation was performed to remove TPH from a diesel contaminated military site. In batch experiments, when 20% H2O2 solution was used for TPH contaminated soil, TPH removal efficiency was 63.5%. Batch experiments investigating biodegradation by adding indigenous microorganisms in pre-H2O2-treated soil were also performed, and TPH removal efficiency of biodegradation was 48.5%, showing an improvement of 19.4% by biodegradation even after chemical oxidation. For a pilot scale feasibility test, a site contaminated with diesel (2.5m×2.7 m×1m) in Korea was selected, and five injection wells and one extraction well were installed in the site. After 0.3 pore volumes of 17.5% H2O2 solution flushing for 15 days, TPH removal efficiency of the site was 51.5%. Seven days after the H2O2 solution flushing was finished, a mixed indigenous microorganism cultured solution (43L) was injected into the wells two times. After the injection of the cultured solution, the average concentration of TPH in the site decreased to 777mg/kg, showing that an additional 19.6% of TPH was removed by biodegradation (total TPH removal efficiency: 71.1%).

Effect of berberine on Escherichia coli, Bacillus subtilis, and their mixtures as determined by isothermal microcalorimetry

The strong toxicity of pathogenic bacteria has resulted in high levels of morbidity and mortality in the general population. Developing effective antibacterial agents with high efficacy and long activity is in great demand. In this study, the microcalorimetric technique based on heat output of bacterial metabolism was applied to evaluate the effect of berberine on Escherichia coli, Bacillus subtilis, individually and in a mixture of both using a multi-channel microcalorimeter. The differences in shape of the power-time fingerprints and thermokinetic parameters of microorganism growth were compared. The results revealed that low concentration (20μg/mL) of berberine began to inhibit the growth of E. coli and mixed microorganisms, while promoting the growth of B. subtilis; high concentration of berberine (over 100μg/mL) inhibited B. subtilis. The endurance of E. coli to berberine was obviously lower than B. subtilis, and E. coli could decrease the endurance of B. subtilis to berberine. The sequence of half-inhibitory concentration (IC(50)) of berberine was: B. subtilis (952.37μg/mL) > mixed microorganisms (682.47μg/mL) > E. coli (581.69μg/mL). Berberine might be a good selection of antibacterial agent used in the future. The microcalorimetric method should be strongly suggested in screening novel antibacterial agents for fighting against pathogenic bacteria.

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45°C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7-8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.

Isolation of halophilic lactic acid bacteria from traditional Chinese fermented soybean paste and assessment of the isolates for industrial potential

Lactic acid bacteria (LAB) from traditional Chinese fermented soybean paste were isolated and identified. A total of 61 LAB were selectively obtained from 32 homemade Chinese soybean pastes. The isolated LAB were divided into two groups by their salt tolerance, 28 halophilic LAB and 33 non-halophilic LAB. Phenotypic analysis showed that these LAB belonged to four genera and 13 species. Tetragenococcus halophilus was the predominant species in the identified strains. Four species of LAB were firstly isolated from fermented soybean food product, Lactobacillus panis, Lactobacillus pentosus, Lactobacillus vaccinostercus and Lactobacillus collinoides. T. halophilus T5 showed vigorous growth and fast acidification in high salt concentration. The volatile compounds of mixed microorganism soybean paste with T. halophilus T5, Zygosaccharomyces and Torulopsis candida, during the different fermentation stage were higher in number than those of normal soybean paste at same processing procedure.

Efficient bio-deodorization of aniline vapor in a biotrickling filter: Metabolic mineralization and bacterial community analysis

A biotrickling filter inoculated with commercial mixed microorganisms B350 was employed to treat N-containing odorous vapor – aniline. Results indicated no aniline could be detected when empty bed residence time (EBRT) was larger than 110s at inlet concentration of 0.30gm−3. The variation of inlet concentration did not change removal efficiencies when concentration is less than 0.21gm−3 at fixed EBRT 110s. Biodegradation mechanism of aniline was tentatively proposed based on identified intermediates and predicted biodegradation pathway as well as final mineralized products. Aniline was firstly biodegraded to catechol, and then to levulinic acid and subsequently to succinic acid. Finally, about 62% aniline carbon was completely mineralized to CO2, while about 91% aniline nitrogen was converted into ammonia and nitrate. Bacterial community in biotrickling filter was found that at least seven bands microbes were identified for high efficiencies of bioreactor at stable state. In all, biotrickling filter seeded with B350 would be a better choice for the purification odorous gas containing high concentration aniline.

Scaled-up bioconversion of fish waste to liquid fertilizer using a 5 L ribbon-type reactor

A scaled-up conversion process of fish waste to liquid fertilizer was performed in a 5 L ribbon-type reactor. Biodegradation was performed by inoculation of autoclaved fish waste with 5.84 × 105 CFU mL−1 of mixed microorganisms for 96 h. As a result, the pH changed from 6.92 to 5.72, the cell number reached 7.28 × 105 CFU mL−1, and approximately 430 g (28.3%) of fish waste was degraded. Analyses indicated that the 96 h culture of inoculated fish waste possessed comparable fertilizing ability to commercial fertilizers in hydroponic culture with amino acid contents of 6.91 g 100 g−1. Therefore, the scaled-up production achieved a more satisfactory fish waste degradation rate (3.61 g h−1) than the flask-scale production (0.24 g h−1). The biodegraded broth of fish waste at room temperature did not undergo putrefaction for 6 months due to the addition of 1% lactate.

Purification of waste gas containing high concentration trimethylamine in biotrickling filter inoculated with B350 mixed microorganisms

A biotrickling filter packed with ceramic particles and seeded with B350 microorganisms was applied to remove trimethylamine (TMA) from gaseous waste. A 100% removal efficiency (RE) was obtained when the empty bed residence time (EBRT) was larger than 110 s at an inlet concentration of 0.30 mg/L. Maximum elimination capacity (EC) was 13.13 g m −3 h −1 (RE = 64.7%) at 55 s of EBRT. TMA concentrations <0.20 mg/L at 83 s of EBRT did not affect the REs (100%). Maximum EC was 13.95 g m −3 h −1 (RE = 78.1%) at a TMA concentration of 0.42 mg/L. Approximately 53.1% of the carbon in TMA was completely mineralized. Bacterial community analysis in the bioreactor revealed more than 21 species in a stable state. Based on all these results, biotrickling filter inoculated with B350 microorganisms is deemed highly capable of ridding waste gas of TMA.

Dietary sulfur concentration has no effect on In Vitro fermentative activity of ruminal mixed microorganisms

We previously reported that elevated concentrations of dietary sulfur (0.65% sulfur, dry basis) in finishing diets containing dried distillers grains with solubles decreased dry matter intake and average daily gains of feedlot cattle. Furthermore, high dietary sulfur concentrations yielded lower ruminal concentrations of volatile fatty acids, but were associated with increased ruminal ammonia concentrations and improved total tract digestibility of the diet. The objective of this study was to investigate, in culture tubes, effects of added sulfur on in vitro dry matter disappearance, volatile fatty acid profiles, and ammonia concentrations from substrates containing different sulfur concentrations when fermented by mixed ruminal microorganisms from a steer fed a diet based on corn and alfalfa.

Comparison of the removal of ethanethiol in twin-biotrickling filters inoculated with strain RG-1 and B350 mixed microorganisms

This study aims to compare the biological degradation performance of ethanethiol using strain RG-1 and B350 commercial mixed microorganisms, which were inoculated and immobilized on ceramic particles in twin-biotrickling filter columns. The parameters affecting the removal efficiency, such as empty bed residence time (EBRT) and inlet concentration, were investigated in detail. When EBRT ranged from 332 to 66 s at a fixed inlet concentration of 1.05 mg L −1, the total removal efficiencies for RG-1 and B350 both decreased from 100% to 70.90% and 47.20%, respectively. The maximum elimination capacities for RG-1 and B350 were 38.36 (removal efficiency = 89.20%) and 25.82 g m −3 h −1 (removal efficiency = 57.10%), respectively, at an EBRT of 83 s. The variation of the inlet concentration at a fixed EBRT of 110 s did not change the removal efficiencies which remained at 100% for RG-1 and B350 at concentrations of less than 1.05 and 0.64 mg L −1, respectively. The maximum elimination capacities were 39.93 (removal efficiency = 60.30%) and 30.34 g m −3 h −1 (removal efficiency = 46.20%) for RG-1 and B350, respectively, at an inlet concentration of 2.03 mg L −1. Sulfate was the main metabolic product of sulfur in ethanethiol. Based the results, strain RG-1 would be a better choice than strain B350 for the biodegradation of ethanethiol.

Effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and nutrient utilization in dairy does

The objective of this study was to evaluate effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and on feed intake and nutrient utilization in dairy does. Batch cultures of mixed rumen microorganisms were used to study effects of different concentrations of fumarate on fermentation with various N sources (ammonia as ammonium bicarbonate, casein amino acids, casein peptides, gelatin peptides) and feeds (bermudagrass hay, mixed diet of 60% bermudagrass hay plus 40% concentrate) for 6 and 24h, respectively. Substrates were grouped into pairs for separate incubations. Monosodium fumarate was added to incubation tubes to achieve final concentrations of 0, 5, and 10mM fumarate. More ammonia accumulated at the end of incubation with added ammonium bicarbonate. Ammonia concentration was higher for peptide compared with amino acid incubation, and for casein peptide compared with gelatin peptide. Addition of fumarate linearly decreased ammonia for all N sources and for feed substrates. For all substrate types, fumarate treatment increased acetate, propionate, and total volatile fatty acids (VFA), decreased acetate to propionate ratio, and tended to reduce branched-chain VFA. Digestion of feed neutral detergent fiber (NDF) by rumen microorganisms was improved by fumarate along with elevated endoglucanase and xylanase activities. In an animal metabolism experiment, 8 dairy does (4 per treatment) were used in a completely randomized design for 21 d. Does were fed a hay plus concentrate diet without (control) or with fumarate (6 g/head per day) supplementation to determine feed intake, whole-tract nutrient digestibility, and N utilization. Fumarate treatment did not affect weight change or feed intake but increased whole-tract digestion of gross energy, crude protein, and cellulose. Digested N was increased by fumarate supplementation; however, N retention was unaffected. Plasma glucose concentration was elevated with fumarate but urea N concentration remained unchanged. Fumarate addition had significant effects on rumen microbial fermentation by decreasing ammonia and branched-chain VFA, and by increasing acetate and propionate, and NDF digestion. These effects were reflected in the improvement in whole-tract gross energy, crude protein, and cellulose digestion and elevated plasma glucose concentration when dairy does were supplemented with fumarate.

Enhanced bio-hydrogen production by anaerobic fermentation of apple pomace with enzyme hydrolysis

A series of batch experiments were conducted to investigate the effects of the HCl-pretreated concentrations, enzyme hydrolysis time and temperature, the cellulase dosage, the ultrasonic time and the fermentation substrate concentration on hydrogen (H 2) production from the anaerobic fermentation of apple pomace (AP). The natural mixed microorganisms from river sludge was used as the seed after being boiled for 15 min. A maximum cumulative H 2 yield (CHYm) of 134.04 ml/g total solid (TS) and an average H 2 production rate (AHPR) of 12.00 ml/g TS/h were obtained from the fermentation of the enzyme-hydrolyzed AAP (a AP soaked with 6% ammonia liquor for 24 h) at the substrate concentration of 15 g/L. The optimal enzyme hydrolysis conditions were proposed as follows: AAP with a cellulase dosage of 12.5 mg/g TS at the hydrolysis substrate concentration of 20 g/L after the ultrasonic irradiation for 20 min was hydrolyzed by the enzyme catalysis at 45 °C and initial pH 5.0 for 48 h.

Applications and integration of chromogenic culture media in clinical microbiology

The ability to rapidly detect and identify micro-organisms is of paramount importance for many microbiology laboratories. The application of new classes of enzymatic-based chromogenic compounds has revolutionised traditional culture media, leading to the developments of a new generation of chromogenic media. Many studies have shown that the chromogenic agar has become more than an isolation media when compared to conventional culture media. A newer, faster, more cost-effective means in the presumptive identification or screening of microorganisms which are either genus, or in some cases species-specific, and allows superior differentiation of mixed micro-organisms in cultures. When supplemented with antibiotics or other agents, chromogenic media allows the screening for multidrug resistance and virulence in many micro-organisms and can be easily integrated with other automated and/or molecular-based methods.

Effect of Sodium Lauryl Sulfate-Fumaric Acid Coupled Addition on theIn VitroRumen Fermentation with Special Regard to Methanogenesis

The aim of the current study was to evaluate the effect of sodium lauryl sulfate-fumaric acid coupled addition on in vitro methangenesis and rumen fermentation. Evaluation was carried out using in vitro gas production technique. Ruminal contents were collected from five steers immediately after slaughtering and used for preparation of inoculums of mixed rumen microorganisms. Rumen fluid was then mixed with the basal diet of steers and used to generate four treatments, negative control (no additives), sodium lauryl sulfate (SLS) treated, fumaric acid treated, and SLS-fumaric acid coupled addition treated. The results revealed that, relative to control, efficiency in reduction of methanogenesis was as follows: coupled addition > SLS-addition > fumaric acid addition. Both SLS-addition and SLS-fumaric acid coupled addition demonstrated a decremental effect on ammonia nitrogen (NH3–N), total short chain volatile fatty acids (SCVFAs) concentrations and the amount of substrate degraded, and an increment effect on microbial mass and microbial yield (YATP). Nevertheless, fumaric acid did not alter any of the previously mentioned parameters but induced a decremental effect on NH3–N. Furthermore, both fumaric acid and SLS-fumaric acid coupled addition increased propionate at the expense of acetate and butyrate, while, defaunation increased acetate at the expense of propionate and butyrate. The pH value was decreased by all treatments relative to control, while, cellulase activity did not differ by different treatments. The current study can be promising strategies for suppressing ruminal methane emissions and improving ruminants feed efficiency.