Inoculum Volume Research Articles

Production of Microbial Biomass Protein by Arachniotus ruber using Banana Peel and its Biological Evaluation in Broiler Chicks

The major issue of the developing countries, i.e. malnutrition, results from intake of diet deficient in proteins. The problem can be resolved by searching for novel and cheap protein sources. Bioconversion of inexpensive fruit waste to an economical product like microbial protein can be useful in the present situation. The aim of the present study was to enhance the crude protein content of banana peels by Arachniotus ruber under solid state fermentation for utilization in broiler feed. Different physicochemical parameters were optimized to increase the yield. Biological evaluation was carried out by replacing 25 and 50% of sunflower meal in broiler feed with the biomass protein. Different parameters e.g.: weight gain, feed efficiency, feed conversion ratio and protein efficiency ratio were recorded. The optimum conditions to achieve highest amount of crude protein (15.8%) on fermentation of banana peels was 1:1.5 substrate water ratio, 2mL inoculum volume, pH 4 after 72 hours of incubation time. Addition of glucose (0.5%), corn steep liquor (0.25%) and MgSO4 (0.05%) enhanced the yield. Outcome of the study revealed that biomass protein produced, can replace up to 50% of the protein supplied by sunflower meal with the positive effect on weight gain of the chicks.

Novel denitrifying bacteria Pseudomonas stutzeri strain D1 - from isolation to the biomass production

An aerobic denitrifier was newly isolated and identified by VITEK? 2 Compact System and MALDI-TOF MS as P. stutzeri strain D1. Sequence amplification indicates that the denitrification genes napA, nirS, norB and nosZ are present in a novel strain D1, as well as in reference strain ATCC 17588. Strain D1 had capability to fully remove 3 g/L of nitrate (as KNO3) in 48 h, while the reference strain completed this task in 60 h. Single factor experiments indicate that the optimal conditions for biomass production were: temperature of 30?C, pH value of 7 and inoculum volume of 5 vol.%. Scaling up of biomass production of both denitrifiers was successfully performed in 3 and 7 L laboratory bioreactors by reaching 9 log CFU/mL of the viable cells. The results demonstrate the feasibility of using investigated P. stutzeri strains in denitrification processes and the simplicity of the up-scaling of biomass production for the treatment of large areas contaminated with nitrate.

Growth Profile of <i>Chaetoceros</i> Sp. and its Steady State Behaviour with Change in Initial Inoculum Size:A Modelling Approach

Monitoring and modelling of the growth profile of microalgae species should be an important tool for the hatchery industries before standardizing the best yielding and cost-effective protocol for their unit. Several factors are responsible in determining the nature of the growth profile. The most important regulator of such growth profile should be the volume of the initial inoculum. In addition, identification and determination of different phases (lag, log, stationary, etc.) of the growth curves of microalgae may be an essential part in the growth profile monitoring. Estimation of growth phases will also help the hatchery scientists in standardizing the commercial culture for industry. Moreover, the transition of different phases can be accurately identified through theoretical models, which are mostly overlooked in simple analysis. Summing up, we have two precise objectives: (1) to study the effects of choice of initial inocula levels on the time to maturity of the Chaetoceros sp., (2) to model the growth profile of the species from which we can theoretically determine its different phases, based on the optical density measurement as a proxy of the biomass. The estimated values of each phase are compared under two initial inocula levels through statistical tests. Using the conceptual approach of the proposed theoretical technique, there is scope for developing a similar model, which can be used in determining cost-effective culture protocol for commercial use.

Biotransformation of eugenol to vanillin by a novel strain Bacillus safensis SMS1003

Due to the extensive applications of vanillin as flavored compound and increasing consumers concern for its natural and environment friendly mode of production, present work was focused on the selection of bacterial isolate capable of producing vanillin using eugenol biotransformation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up to 10.7% (0.055 g/L) at 96 h of biotransformation using growing culture of B. safensis SMS1003 in following culture conditions: eugenol concentration 500 mg/L; temperature 37 °C; initial pH 7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180 rpm. Vanillin was detected as the single metabolite with a molar yield of 26% (0.12 g/L) at 96 h using resting cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and mass spectroscopy.

An improved carbapenem inactivation method, CIMTrisII, for carbapenemase production by Gram-negative pathogens.

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa. We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae, Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

166 Effect of gas production recording system and inoculum volume on in vitro fermentation of corn distillers dried grains with solubles (cDDGS) or soybean hulls using pig fecal inoculum.

166 Effect of gas production recording system and inoculum volume on in vitro fermentation of corn distillers dried grains with solubles (cDDGS) or soybean hulls using pig fecal inoculum.

Evolutionary Prediction of Biohydrogen Production by Dark Fermentation

The present work is a study of the performance and effect of operational parameters on biohydrogen production from palm oil mill effluent by dark fermentation in batch mode. The process parameters examined are pH (5, 5.5, and 6), temperature (30, 35, and 40 °C), substrate concentration (5000, 12 500, and 20 000 mg L−1) and inoculum volume (20, 25, and 30 mL). The inoculum concentration prepared was 10 000 mg L−1 volatile suspended solids. The experiments were designed by response surface methodology (RSM). The highest chemical oxygen demand (COD) removal, hydrogen percentage (H2%) and hydrogen yield (HY) obtained were 58.3%, 80%, and 4.83 mol H2/mole of COD consumed, respectively. Based on the experimental data obtained with the RSM design, gene expression programming (GEP) was developed to predict the COD removal, hydrogen production, and hydrogen yield as process responses. The projected models were assessed based on the correlation coefficient (R2), root mean square error, mean absolute relative error, scatter index, and BIAS. The results demonstrate that the GEP model outperformed the RSM model and was superior in predicting the response variables. Partial derivative sensitivity analysis was also employed to assess the effect of each variable on COD%, H2%, and HY prediction. The prediction uncertainty for COD%, H2%, and HY was quantified, and the results were ±0.11, ±0.17, and 0.015, respectively. According to the results, the GEP model is more efficient than the RSM model in predicting the experimental data for biological hydrogen production in the dark fermentation process.

Xanthan gum biosynthesis using Xanthomonas isolates from waste bread: Process optimization and fermentation kinetics

Waste bread hydrolysate was used as a biosource for xanthan production by various isolates and standard bacteria (Xanthomonas campestris DSM 19000). Influence of operational conditions used in the process of xanthan production were evaluated through yield of xanthan, rheological properties of aqueous solution and fermentation kinetics. For the highest yield and viscosity, optimum conditions including carbon source concentration, inoculum volume and agitation rate were determined for 4 different strains by using response surface methodology. The highest gum yield as 14.3 g/L was obtained by Xanthomonas axonopodis vesicatoria and the highest conversion rate of waste bread to xanthan gum was found as %14.1 for Xanthomonas hortorum pv. pelargonii. Whereas, the highest aqueous solution viscosity of gum produced from standard bacteria was 11.2 Pa.sn at glucose ratio of 4%, inoculum volume of 5% and mixing rate of 225 rpm. For fermentation kinetics; the values of growth associated parameters revealed that they are mostly affected by the rate of xanthan gum production and substrate consumption. In general, optimum conditions to obtain the highest xanthan gum yield were different from that to achieve the highest viscosity. This study shows the potential of waste bread hydrolyzates as the promising economic carbon source for xanthan gum production.

Development and application of a higher throughput RSV plaque assay by immunofluorescent imaging

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent “objects”. The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.

Optimization of submerged cultivation parameters for a novel biological pretreatment of rice straw to enhance enzymatic hydrolysis

Statistical optimization of submerged cultivation parameters was carried out by Taguchi orthogonal array design for delignification of rice straw using a novel alkalophilic fungus MVI.2011. Parameters such as media composition, pH, temperature, inoculum volume, biomass volume and incubation time were optimized in this study. The objective was to maximize the release of glucose (% of saccharification) at the end of 8 h enzymatic hydrolysis. Incubation time and concentration of peptone in the cultivation media, with P‐values 0.027 and 0.041, respectively, were the most significant factors affecting the delignification reaction. Optimal run gave percentage of saccharification of 14.18 ± 1.13% (g g−1) analogous to an increase of 1.96‐fold in comparison with untreated rice straw (7.23 ± 0.04% [g g−1]). The reaction was scaled up to delignify 120 g rice straw in a 5‐L fermenter, the % saccharification of which was 13.5 ± 0.68% (g g−1), equivalent to an increase of 1.87‐fold when compared to untreated straw. Fungal pretreatment with MVI.2011 exhibited exemplary hydrolysis rate and demanded less than a week for delignification. The study reports for the first time, a biological pretreatment technique that takes only 6 days. This method can be extended to the pretreatment of other lignocelluloses. © 2018 American Institute of Chemical Engineers Environ Prog, 38:e13020, 2019

Bioconversion of Agricultural Waste to Butyric Acid Through Solid State Fermentation by Clostridium tyrobutyricum

The present study was aimed to develop a successful bioprocess through solid state fermentation (SSF) technique for the butyric acid production using agriculture wastes like wheat bran, rice polishings and molasses anaerobically by Clostridium tyrobutyricum DSM 2637. Different physiochemical parameters like substrate: water ratio, inoculum volume, different ionic salts concentrations, an additive effect of molasses and corn steep liquor were optimized to boost the butyric acid production through SSF of the growth medium anaerobically by Clostridium tyrobutyricum DSM 2637 at 37 °C, pH 6.0. The fermentation of the growth medium was terminated after 72 h of incubation. Quantification of butyric acid was done by Deniges method. The growth medium containing rice polishings, molasses, and wheat bran when subjected to determine substrate: water ratio through SSF at 37 °C, pH 6.0 anaerobically by Clostridium tyrobutyricum DSM 2637 yielded 5.63, 5.5, 4.26 mg/100 g of butyric acid respectively. The present investigation demonstrated that rice polishings supplemented with corn steep liquor is a good agricultural waste for butyric acid production. The SSF technique can efficiently and successfully be utilized for better yield of butyric acid on the pilot scale.

Dye-tolerant marine Acinetobacter baumannii-mediated biodegradation of reactive red

The objective of this study was to isolate a potent dye-degrading microbe that can be used to reduce the pollution caused by industrial dyes. Reactive red 198 is an extensively used textile dye and is a major environmental pollutant in water bodies. In this study, a bacterial strain was isolated from sea sediments and identified as Acinetobacter baumannii with 16S rRNA sequencing. The isolated bacteria were immobilized in calcium alginate and decolorization studies were carried out to determine the optimum pH, temperature, dye concentration, inoculum volume, and static/agitated condition using the one factor at a time (OFAT) approach. The Box-Behnken design, a type of response surface methodology, was adopted to improve the degradation efficiency. At 37°C using an inoculum volume of six beads, 96.20% decolorization was observed in 500 mg/L of reactive red 198 after 72 h. Dye degradation was confirmed with UV-visible spectroscopy and Fourier-transform infrared (FTIR) spectroscopy studies of the dye and degraded metabolites. Microbial toxicity studies using Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and phytotoxicity studies using Vigna radiata proved that the toxicity of the dye was significantly reduced after degradation. We can conclude that the isolated A. baumannii strain is an efficient dye-degrading microbe that can be used to reduce the pollution caused by industrial dyes.

Enhanced halophilic lipase secretion by Marinobacter litoralis SW-45 and its potential fatty acid esters release.

An approach was made to enhance the halophilic lipase secretion by a newly isolated moderate halophilic Marinobacter litoralis SW-45, through the statistical optimization of Plackett-Burman (PB) experimental design and the Face Centered Central Composite Design (FCCCD). Initially, PB statistical design was used to screen the medium components and process parameters, while the One-factor-at-a-time technique was availed to find the optimum level of significant parameters. It was found that MgSO4 · 7H2 O, NaCl, agitation speed, FeSO4 · 7H2 O, yeast extract and KCl positively influence the halophilic lipase production, whereas temperature, carbon source (maltose), inducer (olive oil), inoculum size, and casein-peptone had a negative effect on enzyme production. The optimum level of halophilic lipase production was obtained at 3.0 g L-1 maltose, 1% (v/v) olive oil, 30 °C growth temperature and 4% inoculum volume (v/v). Further optimization by FCCCD was revealed 1.7 folds improvement in the halophilic lipase production from 0.603 U ml-1 to 1.0307 U ml-1 . Functional and biochemical characterizations displayed that the lipase was significantly active and stable in the pH ranges of 7.0-9.5, temperature (30-50 °C), and NaCl concentration (0-21%). The lipase was maximally active at pH 8.0, 12% (w/v) NaCl, and 50 °C temperature. Besides, M. litoralis SW-45 lipase was found to possess the promising industrial potential to be utilized as a biocatalyst for the esterification.

Enzymatic delignification and saccharification of Bambusa bambos for biobutanol production

Conversion of lignocellulosic biomass to energy is a transformational approach for addressing the escalating energy demand and environmental concerns along with waste management. In the present study, bamboo rich in holocellulose content (cellulose, 42.47 ± 2.3% (w/w) and hemicellulose, 20.10 ± 1.0% (w/w)) was used for biobutanol production. A concoction of laccases (as ligninolytic enzyme) and saccharolytic enzymes (cellulases and xylanases) were used in a single vessel for reducing sugar production from bamboo. Bamboo hydrolysate had glucose, xylose and cellobiose as reducing sugars which were used by Clostridium beijerinckii (ATCC 55025-E604) for ABE (acetone, butanol, and ethanol) production. C. beijerinckii utilized glucose, xylose and cellobiose efficiently and produced ABE when commercial sugars were studied individually. Process parameters studies showed that ABE yield was 0.35 g/g-sugar at 37 °C, pH 6.5, inoculum volume 12% v/v, inoculum age 18 h and fermentation time 60 h. Further optimization of C/N ratio, methyl viologen and CaCO3 Led to increase in the yield of butanol by 30.39%, w/v while the overall ABE yield was increased by 17.79%, w/v as compared to control (without use of C/N ratio, methyl viologen and CaCO3). The enzymes used for these studies are of high titer and produced indigenously utilizing cheaper substrates such as agroresidues under solid state fermentation. Moreover, overall process effectiveness is promising and attractive because of higher conversion efficiency, eco-friendliness and biodegradable nature.

Optimization of culture conditions for enhanced lipase production by an indigenous Bacillus aryabhattai SE3-PB using response surface methodology

Lipases are enzymes that hydrolyze fats into fatty acids and glycerol at the water–lipid interface and are also involved in a variety of bioconversion reactions in non-aqueous and micro-aqueous environments. In this study, we optimized the culture conditions for extracellular lipase production by an indigenous lipase-producing bacterial strain isolated from lipid-rich wastewater, using response surface methodology. The studied isolate was identified as Bacillus aryabhattai SE3-PB by polymerase chain reaction and analysis of 16S rDNA. Sunflower oil was found to induce maximum lipase production. Face centered central composite design revealed that temperature (40 °C), pH (7.6), inoculum volume (2.8%, v/v), agitation (193 rpm) and inducer oil concentration (2%, v/v) significantly influenced lipase production at the respective optimum conditions. The coincidence of observed lipase production (264.02 ± 1.94 U/mL) with predicted lipase yield (265.82 U/mL) coupled with a high correlation coefficient (R2 = 0.9919, P < 0.01) confirmed the validity of the model. A 7.2-fold increase in lipase production was obtained in the optimized medium compared to the basal medium. These findings provide the first report on lipase production and optimization by B. aryabhattai SE3-PB and suggest a rational choice of optimum processing conditions for commercial lipase production by B. aryabhattai SE3-PB.

Enhanced production of butyric acid by solid-state fermentation of rice polishings by a mutant strain of Clostridium tyrobutyricum

Purpose: To enhance butyric acid production by solid-state fermentation with a hyper-producing mutant of Clostridium tyrobutyricum generated by random mutagenesis.Methods: Wild type C. tyrobutyricum was mutagenized with UV irradiation, nitrous acid, and ethidium bromide to obtain a hyper-producing strain. Various physiochemical parameters were optimized to increase the butyric acid yield.Results: The UV-induced mutant (C.TUV) produced significantly higher concentrations of butyric acid than the wild type parent, nitrous acid-induced, and ethidium bromide-induced strains. C.TUV increased butyric acid production 1.4-fold more than the parent strain. Fermentation with C.TUV with 2.5 g of rice polishings (w/w), a 2 % inoculum volume (v/v), and a 48-h incubation period at 37°C under anaerobic conditions produced 11.63 mg/100 g of butyric acid. The addition of 0.6 % corn steep liquor as a nitrogen source increased the butyric acid concentration to 26.09 mg/100 g.Conclusion: These optimized fermentation parameters on a small scale can be used on a commercial scale to mass-produce butyric acid.Keywords: Butyric acid, Mutant, Clostridium tyrobutyricum, Mutagen, Solid-state fermentation

Direct chromatographic study of the enantioselective biodegradation of ibuprofen and ketoprofen by an activated sludge

The quantification of the enantiomeric fraction (EF) during the biodegradation process is essential for environmental risk assessment. In this paper the enantioselective biodegradation of ibuprofen, IBU, and ketoprofen, KET, two of the drugs most consumed, was evaluated. Biodegradation experiments were performed in batch mode using a minimal salts medium inoculated with an activated sludge (collected from a Valencian Waste Water Treatment Plant) and supplemented with the racemate of each compound. The inoculum activity was verified using fluoxetine as reference compound. The experimental conditions used (analyte concentration and volume of inoculum) were chosen according to OECD guidelines. In parallel, the optical density at 600 nm was measured to control the biomass growth and to connect it with enantioselectivity. Two RPLC methods for chiral separations of IBU and KET using polysaccharides-based stationary phases were developed. Novel calculations and adapted models, using directly the chromatographic peak areas as dependent variable, were proposed to estimate significant parameters related to the biodegradation process: biodegradation (BD) and EF values at given time, half-life times of (R)- and (S)-enantiomers, number of days to reach a complete BD and the minimum EF expected. The modelled BD and EF curves fitted adequately the data (R2 > 0.94). The use of these new equations provided similar results to those obtained using concentration data. However, the use of chromatographic peak areas data, eliminates the uncertainty associated to the use of the calibration curves. The results obtained in this paper indicate that an enantiorecognition towards IBU enantiomers by the microorganisms present in the activated sludge used in this study occurred, being the biodegradation of (R)-IBU higher than that of (S)-IBU. For KET, non-enantioselective biodegradation was observed.

Pretreatment of organic composite waste mixtures for enhanced biomethanantion

ABSTRACTHydrolysis is the rate-limiting step for the anaerobic digestion of wastewater. overcome this rate-limiting step,​ pretreatment of organic waste is required. The objective of the present study is to find the effects of various pretreatment parameters along with their optimal values to make the process economically feasible. A full factorial design was used to design the experiments with temperature, duration of heat treatment, pH, and the volume of inoculum added as independent variables and cumulative methane volume (CMV) as the dependent variable. Residual curve shows that the errors are having a constant variance. Contour plots and three-dimensional surface plots show variation in the response variables. Among the independent variables, inoculum has the most significant effect on the degradation process followed by pH and temperature. Duration of heat treatment induces little effect on biogas yield. The interaction terms have a significant effect on the CMV.

내산성이 우수한 Acetobacter pasteurianus AFY-4 균주를 이용한 다래 식초 초산발효 최적화

Acetobacter pasteurianus AFY-4 was isolated from fermented vinegar in Hongcheon-gun, Gangwon-do. The A. pasteurianus AFY-4 strain had a higher tolerance to low-pH. Optimization of acetic acid fermentation of hardy kiwi (Actinidia arguta) was conducted using the A. pasteurianus AFY-4 strain based on the response surface methodology (RSM). The optimization of acetic acid fermentation for producing hardy kiwi vinegar was determined by five levels of initial ethanol, initial acetic acid, and acetic acid bacteria (AAB) inoculum volume, using the central composite design. The acetic acid yield and residual ethanol for acetic acid fermentation of hardy kiwi optimized by the RSM were significantly different. The optimal conditions for acetic acid fermentation of hardy kiwi vinegar were 8.95% of the initial ethanol, 1.86% of the initial acetic acid, and 25.02% of the AAB inoculum volume.

Characterization and optimization of a heterotrophic bacterium for sulfide degradation

The emission of hydrogen sulfide in the waste gas from slaughter plant, fishmeal feed processing and some other food industrial processing could cause serious air pollution to the surrounding environment. The purpose of this study was to screen heterotrophic bacterium strains for the removal of hydrogen sulfide odor. One heterotrophic bacterial mutant ZJNB-B3 was derived from the sulfide degrader Bacillus cereus XJ-2 and its sulfide removal efficiency was 97%. Based on the morphology studies, biochemical tests and 16S rRNA gene analysis, the strain was identified as Bacillus cereus ZJNB-B3. The NCBI GenBank accession number is MF679650. Batch tests showed that the strain tolerated up to 300 mg/L of toxic S²⁻ concentration. Response surface methodology was applied to optimize the conditions of degradation of sulfide. The optimal parameters were as follows: initial sulfide concentration 211.8 mg/L, initial pH 6.72, inoculum volume 5.04%, and incubation temperature 30 ℃. The accumulated sulfate concentration was 63.8 mg/L and the sulfide removal efficiency was 97.3% after 48 h incubation. No sulfuric acid was generated during sulfide oxidation by the strain. Sulfide could be removed effectively by this strain under mild pH conditions. The results suggested that the strain may have great industrial application potential. This study provides the fundamentals for the removal of hydrogen sulfide gas.