Inoculum Volume Research Articles

Increased production of alkaline polygalacturonate lyase in the recombinant WB43CB by optimizing the medium

Alkaline polygalacturonate lyase (PGL), which was the central enzyme of green environmental enzyme treatment technology, has been widely used in textile, papermaking and beverage production. In the previous work, Bacillus subtilis was used as the expression host, and the PGL gene derived from B. subtilis WSHB04-02 was successfully expressed in the engineered strain WB43CB by regulating of molecular elements such as signal peptide, promoter and SD sequence. In this study, the fermentation medium was optimized from four aspects of carbon source, nitrogen source, liquid volume and inoculum volume, and the extracellular PGL enzyme activity in WB43CB increased from 264.5 U·mL-1 to 461.96 U·mL-1, which laid a solid foundation for the industrial production of PGL.

Optimization of fermentation conditions for the production of acidophilic β-glucosidase by Trichoderma reesei S12 from mangrove soil

Vegetative biomass contains a large number of macromolecular substances such as cellulose and hemicellulose, which can be used by microorganisms to produce biofuels and other chemical byproducts. In this study, a filamentous fungus with high β-glucosidase activity was isolated from mangrove soil in Hainan Province, China. Through morphological identification and internal transcribed spacer (ITS) sequence analysis, the strain was identified as Trichoderma reesei. Fermentation conditions were optimized with the response surface method to improve β-glucosidase activity. Inoculum, pH and liquid volume in flask were found to be the key factors. We further examined the optimal range of the three factors using steepest ascent path, and optimum conditions were further investigated according to a Box-Behnken design. We calculated the optimized fermentation conditions to be: carbon source (microcrystalline cellulose (MCC)) 1%, nitrogen source (yeast extract) 0.5%, pH 3.68, inoculum of 3.22 × 105 cfu/mL, temperature 28 °C, shaking speed of 160 rpm, and liquid volume in flask of 84.74 mL/250 mL. These conditions increased the β-glucosidase activity 6-fold to 1.13 U/mL compared to that before optimization. The resulting β-glucosidase had an optimum pH of 5.0 and an optimum temperature of 45 °C, and showed high thermal stability at 30-60 °C.

Characterization of Wet Mesophilic Biomethanization of Three Types of Materials: Chicken Dung, Rabbit Poop and Pig Slurry

Biomethanization is a process leading to the production of biogas. Characteristics effects of some materials on biomethanization results are not well known by now. That is the raison of studying the effect of the chemical composition of chosen substrates that are chicken dung, pig slurry and rabbit poop on biomethanization characteristics. These substrates of 1 mm particles size and 13.33% water content were first subjected to chemical analysis. Experimentation consisted of mixing 1.3 kg of each substrate with 6.2 L of water for a 15% dry matter content in the final mixture. Biomethanized cow dung (0.81 L) was added as inoculum to each mixture to give a ratio of inoculum volume to mixture volume of 10%. The biomethanization temperature was maintained at 38°C during all the process. The evolution of the composition and the biogas yield of each substrate was monitored using respectively an infrared biogas analyser and a digital manometer installed on each experimental unit. The main results were as follows: the C/N ratio was highest in rabbit poop (28.57), followed by pig slurry (14) and finally chicken dung (11). The organic matter content was also highest in rabbit poop (80%), but followed by chicken dung (65%) and pig slurry (50%). The final methane content was highest in rabbit poop (58.61%), followed by chicken dung (51.59%) and pig slurry (50.83%). The final percentage of carbon dioxide was highest in the pig slurry (12.62%), followed by the rabbit poop (11.31%) and finally the chicken dung (9.98%). In terms of biogas yield and hydraulic retention time, rabbit poop gave the highest yield of 0.109 m³.kg^-1 of dry matter in 37 days. This were followed by chicken dung with 0.067 m³.kg^-1 of dry matter in 27 days and pig slurry with 0.037 m³.kg^-1 of dry matter in 20 days. In the light of these results, the main conclusion is that, more the organic matter content is high and C/N ratio is in the optimal range of 25 to 30, higher are biogas yield and methane content, and longer is the hydraulic retention time.

Statistical optimization of cultural parameters for the optimized production of alginic acid using apple (Malus domestica) peels through solid-state fermentation

The industrially significant biopolymer, alginic acid, is mainly obtained from farmed brown seaweeds. Owing to differences in structural composition of alginate isolated from various species, the bacterial alginate has gained significant importance. Pakistan meets the industrial demand of alginate by importing it from developed countries. The current research was planned to assess the potential of apple peels (Malus domestica) for alginate production by using Azotobacter vinelandii through solid-state fermentation. Various cultural parameters were optimized using CCD of response surface methodology (RSM) like incubation days, pH, temperature and inoculum volume. Maximum yield of alginate (180.64 mg/gds) was achieved by fermentation of apple peels at substrate water ratio of 1:3 for 2 days with 3 mL volume of inoculum at pH 7.5 and 37.5 °C. Statistical analysis showed the F value and p value as 10.93 and 0.00 that indicates that the proposed model was highly significant. The goodness of fit of the model was determined by a coefficient of determination (R2) having a value of 90.5% indicating the accuracy of the model. Alginate produced was quantified by HPLC method and was found to be 97% pure in contrast to standard (100% pure of Sigma-Aldrich). The results indicated that alginate can be efficiently produced through solid-state fermentation by utilizing cheap fruit by-product as the substrate.

Statistical optimization for the efficacious degradation of reactive azo dyes using Acinetobacter baumannii JC359

The aim of this study is to biodegrade the reactive azo dyes- Reactive black 5 (B-GDN), Reactive red 120 (RP) and Reactive blue 19 (RNB) using bacteria Acinetobacter baumannii JC359. Optimization of the process variables such as pH, temperature, dye concentration, incubation time, inoculum volume and dynamic incubating conditions for dye decolorization were performed using One Factor At a Time (OFAT) approach. Box- Behnken Design (BBD) of Response Surface Methodology (RSM) was further used to optimize the process variables. Decolorization rates of 98.8% for B-GDN, 96% for RP and 96.2% for RNB were observed after treating with A. baumannii for 48 h using the obtained design value. UV–Visible spectrophotometry and FT-IR spectral scan of dye and degraded metabolites confirmed that biodegradation had taken place. Further, the phytotoxicity evaluation was performed with Vigna radiata seeds and the degraded metabolites proved to be non-toxic. Docking studies were performed and it was found that there was significant binding affinity between the dyes and azoreductase enzyme of A. baumannii. Thus, the biodegradation of these reactive azo dyes was found to be a suitable alternative for the effective treatment of textile dyes.

Effect of cultural conditions on the growth and linamarase production by a local species of Lactobacillus fermentum isolated from cassava effluent

BackgroundThis study was designed to investigate the effect of cultural conditions on growth and production of linamarase by a local species of Lactobacillus fermentum isolated from cassava effluent. Isolation and identification of bacteria from cassava effluent were carried out using the culture-dependent method and polyphasic taxonomy, respectively, while screening for cyanide degradation, and the effects of cultural conditions on the growth and linamarase activity of L. fermentum were investigated based on standard procedures.ResultsA total of twenty-one bacterial isolates were obtained from cassava effluent, and isolate MA 9 had the highest growth of 2.8 × 1010 cfu/ml in minimum medium, confirmed as safe, identified as Lactobacillus fermentum and selected for further study. The highest growth of 2.498 OD and linamarase activity of 2.49 U/ml were observed at inoculums volume of 0.10 ml at 48-h incubation period, while optimum growth of 1.926 OD and linamarase activity of 1.66 U/ml occurred at pH 5.5. At 37 °C, the optimum growth of 0.34 OD was recorded with the highest linamarase activity of 0.81 U/ml at 30 °C.However, the incubation period of 48 h stimulated an optimum growth of 3.091 OD with corresponding linamarase activity of 1.81 U/ml, while the substrate concentration of 400 ppm favours a maximum growth of 2.783 OD with linamarase activity of 1.86 U/ml at 48 h of incubation. The supplemented of 10 mM calcium ions stimulated optimum linamarase activity of 2.65 U/ml.ConclusionLactobacillus fermentum could be used as starter culture in cassava fermentation for the production cassava-based product with reduced cyanide content.

Rat-Bite Fever: Updated Recommendations For Culture And Isolation Of Streptobacillus Moniliformis Using An Automated Continuous Blood Culture Instrument In A Clinical Setting

Abstract Introduction/Objective Rat-bite fever and Haverhill fever are difficult to diagnose in a clinical setting due mostly to clinicians and laboratory professionals being unable to culture the causative agent-Streptobacillus moniliformis. SPS in blood culture bottles has historically been implicated as the complicating factor. Methods Utilizing the BDFX40 automated continuous blood culture bottle system and novel quantitative PCR data, we present how blood volume is critical in order to consistently detect, isolate and grow the organism in the presence of SPS using modern laboratory instrumentation in a clinical setting. Results We demonstrate here that 10ml of blood was determined to provide optimal results for detection and growth of S. moniliformis in 0.05% SPS. For all isolates tested, 100% (n=56) were detected or alerted as positive by the instrument, with the longest time required for detection being 102 hours (n=1) and the fastest time to detection being recorded at 13.4 hours. (n=1) with an average time of 26.5 hours (n=56). Conclusion During the course of this study, we determined that blood inoculum volume played a significant role in organism growth and detection. We found that in 100% of the isolates tested (and all the variations of testing within), SPS (up to a concentration of 0.05% w/v) in blood culture media appeared to be counteracted, allowing for the growth detection and culturing of S. moniliformis using an automated continuous blood culture system when 10ml of blood was used as an inoculum. This is the first study to report and suggest that a specific blood volume is critical when utilizing a closed commercial blood culture system to detect S. moniliformis, this research is the largest study of Streptobacillus moniliformis isolates to date.

Screening and formulation of culture conditions for pyruvate production by candida glabrata MCC 1800 from corn steep liquor

Eleven culture parameters were screened by the Plackett–Burman design for pyruvate production to develop a standard protocol for media formulation. In an incubator shaker, the screened optimal conditions for pyruvate production were: carbon source 24.642g/L, nitrogen source 11.89 g/L, KH2PO4 1.185 g/L, thiamine 13.302 µg/L, biotin 13.927 µg/L, CaCO3 43.432g/L, MgSO4•7H2O 0.346 g/l, inoculum age 3 days, inoculum volume 6% (v/v), agitation 220 rpm, incubation time 46.412 h and incubation temperature 34.582℃. Except CaCO3 all factors were significant and agitation was considered a dummy factor in the experiment.

Degradation and detoxification of leather tannery effluent by a newly developed bacterial consortium GS-TE1310 for environmental safety

The untreated/partially treated effluent discharged from leather tanning industries is heavily polluting our water and soil resources. Hence, the adequate treatment/detoxification of tannery effluent (TE) is required before its safe disposal into the environment. In the present study, an effective degradation of real TE was attained by a newly developed bacterial consortium GS-TE1310 within 120 h with 76.12, 85.32, 71.89, 48.59, 78.81, 69.53, 71.22, and 88.70 % reduction in pollution parameters such as COD, BOD, TDS, phosphate, sulphate, nitrate, Cr, and phenol, respectively. The HP-LC, FT-IR, and GC–MS study showed that most of the organic contaminants identified in the untreated TE were completely mineralized/degraded into new degradation products in the treated TE by the newly developed bacterial consortium GS-TE1310 at 7 pH, 0.5 % glucose and ammonium chloride, 120 rpm, and 20 mL inoculum volume. Further, the bacterially treated TE was used for the phytotoxicity evaluation using Phaseolus aureus L as a terrestrial model organism. Results revealed that the toxicity of bacterially treated TE was significantly reduced, allowing the 70 % germination of seeds, and thus, confirmed the detoxification of leather TE. Overall, the newly developed bacterial consortium GS-TE1310 demonstrated a remarkable potential to efficiently treat/detoxify leather TE for environmental safety.

An Ecological and Miniaturized Biological Method for the Analysis of Daptomycin Potency

Physicochemical and microbiological methods are found in the literature for the analysis of daptomycin, an antimicrobial. This paper brings a miniaturized turbidimetric microbiological method for analysis of daptomycin in lyophilized powder. The method was performed using 96-well microplates, 4-h incubation, 2, 4 and 8 μg/mL, 7% Staphylococcus aureus ATCC 6538 IAL 2082, and BHI broth. Linearity was proven by obtaining analytical curves with a correlation coefficient greater than 0.99 and statistical evaluation by ANOVA. The method was also selective, since the standard and sample analytical curves were parallel, proving that the excipient does not interfere with daptomycin analysis. Intraday, interday and inter-analyst precision presented RSDs of 2, 2.27, and 1.08%, respectively. Accuracy was assessed by the recovery test, where known quantities of standard solution are added to the sample and an average recovery value of 100.73% (RSD = 0.71%) was obtained. The present method was robust when minor changes were made in the parameters of used antimicrobial volume, inoculum volume and incubation time. This work is an innovative and ecological proposal and has advantages such as (i) less waste generation, (ii) miniaturized quantities of sample, culture media and inoculum, (iii) no need to use formaldehyde as in the traditional turbidimetric method, (iv) lower volume of glassware used and (v) shorter incubation time compared to other methods as agar diffusion requiring approximately 24 h. This work is focuses on a current, innovative and sustainable theme for pharmaceutical analysis around the world.

Optimization of microbiological plastic film test plate conditions for rapid detection of antibiotics in milk

Food safety and quality are issues of great concern to food producers and consumers. Key challenges towards achieving these include availability of rapid, user-friendly, economic and reliable techniques to detect problems such as antibiotic residues. Therefore, the present study was conducted to evaluate performance of plastic film test plate (PFTP) as influenced by antibiotic type, test bacterium, inoculum volume and concentration. Comparison was made to the microtiter test plate (MTP) and the National Standard method (SN/T 3979-2014). For this, antibiotic susceptibility testing was conducted on four bacterial types (Micrococcus luteus, Streptococcus thermophillus suspensions, Staphylococcus aureus and Coliform bacteria) against three antibiotics (penicillin G, sulfadiazine and tetracycline). Results showed high susceptibility of Micrococcus luteus to penicillin G with minimum inhibitory concentration of 3 µg L−1 and 1 µg L−1 via PFTP and MTP respectively. Optimum performance was realized at bacterial concentration of 104 CFU ml−1 with detection limit of 1 µg L−1, sensitivity and predictive positive value (PPV) of 81–83% and 97–100% respectively. Detection time was recorded as 6 and 9 h for MTP and PFTP respectively compared to the 18–24 h of the National Standard method (SN/T 3979–2014). The microbiological plastic film test plate under optimized bacterial culture conditions demonstrated tremendous potential for rapid and reliable detection of antibiotics in milk.

Optimisation of fungal glucoamylase production by Response Surface Methodology and characterisation of the purified enzyme

A statistical approach was made for the production of glucoamylase by Aspergillus niger using wheat bran and green gram as fermentation medium. Box-Behnken design used to analyse the simultaneous impact of the substrate, hydration, inoculum volume, fermentation time and temperature, using a second-order polynomial. The experimental results were in good agreement with the proposed regression model with R2 = 0.9322 (p<0.05). The maximum yield (313 U/gds against the predicted value of 306 U/gds) was obtained using 12 gm substrate, 55% moisture, 1 gm sucrose and 0.05 gm tryptone. The purification and characterisation of the enzyme were also studied. Optimum thermal and pH stability was 60 °C and 5 respectively.

Bioaccumulation of Ni(II) on growing cells of Bacillus sp.: Response surface modeling and mechanistic insight

Abstract The growing cells of Bacillus cereus M 16 1 , depending on the growth phase of culture was found to bioaccumulate Ni(II) from its aqueous solution to the extent of 80% wherein surface binding ( ∼ 60%) dominated over intracellular accumulation ( ∼ 20%). No growth was observed in the metal ion concentration beyond 50 mg L − 1 . The hydroxyl, amine, carboxylate and phosphate groups of cell surface were the most important moieties in chemical interactions with Ni(II) ions as measured by FTIR. The surface characteristics of cells were investigated meticulously by FE-SEM equipped with EDXA indicating conspicuous changes due to metal accumulation. The potential effects of the process variables [initial pH (A), temperature (B), inoculum volume (C) and medium volume (D)] were optimized using Response Surface Methodology (RSM) followed by Box–Behnken design to study the bioaccumulation phenomenon. The maximum Ni(II) accumulation was noted at 6.5, 32.5 °C, 2.5% and 50 mL corresponding to pH, temperature, inoculum volume and medium volume, respectively. The most significant factors including one independent variable (D) and two quadratic effects (B2 and D2) were identified by ANOVA. The experimental response under these optimum conditions was found to be 78.76% which closely matched the yield (78.52%) predicted by the statistical model with R 2 = 0 . 8790 .

Biotransformation of Isobutyraldehyde to Isobutanol by an Engineered Escherichia coli Strain

Introduction: Biotransformation process has been used in various industries due to its ability to produce valuable chemicals and address environmental concerns. Propylene hydroformylation is a process in which n-butyraldehyde and isobutyraldehyde are produced. N-butyraldehyde is a high valuable chemical with many industrial applications, while isobutyraldehyde produced as a by-product is an environmental pollutant. This study offers a biotechnological approach for conversion of isobutyraldehyde into a high-value substance. An engineered strain of Escherichia coli was developed by genomic insertion of alcohol-dehydrogenase gene (adhA) from Lactococcus lactis which can convert isobutyraldehyde into isobutanol.Materials and Methods: The adhA gene was engineered to substitute some of its amino acids to result in a more efficient enzyme. Engineered gene was synthesized and introduced into E. coli genome to develop recombinant E. coli EG-296 strain. In addition, by using the Qualiteck-4 software, 16 well-defined experiments (L16 Orthogonal array) with two levels of seven variable parameters were used to optimize the process efficiency.Results: The findings of this study revealed that the E. coli strain EG-296 is capable of converting isobutyraldehyde into isobutanol. The optimization results showed that optimum medium composition for the highest isobutanol production were 10 g/L glucose or glycerol as carbon source, 10 g/L NH4CL as nitrogen source, mid-log of inoculum age, and 1% inoculum volume in 25ml medium. After optimization, 560 mg/L isobutanol was produced from 600 mg/L isobutyraldehyde with 91% yield.Conclusions: Recombinant E. coli strain with a relatively optimum medium can be used to remove isobutyraldehyde in refineries or other industries producing this chemical as a by-product.

Box-Behnken Design–Directed Optimization of Wickerhamomyces anomalus–Mediated Biotransformation Process to Enhance the Flavonoid Profile of Polyherbal Extract

The present study explored biochemical transformation and optimization of fermentation method for enhanced content of flavonoids in a composite extract of Syzygium cumini seed, Mangifera indica seed, Momordica charantia fruit, and Gymnema sylvestre leaf along with the fermentation kinetics. The herbs were mixed and extracted in hot water. The extract was fermented using Wickerhamomyces anomalus (yeast). Box-Behnken design was applied to determine the best possible combination of variables to obtain the maximum content of flavonoids. The variables were substrate concentration (20%, 40%, and 60%), inoculum volume (2.5%, 5%, and 7.5%), and incubation time (60 h, 72 h, and 84 h). Further, the physical parameters during fermentation were determined to establish the kinetics of the process. The content of Luteolin, Quercetin, Kaempferol, and Isorhamnetin was positively affected by all three variables whereas Apigenin content was negatively affected by incubation time, i.e., the higher the incubation time, the lower the content of Apigenin. Overall content of flavonoids was affected by interactions between variables as indicated by contour plots. Actual content of flavonoids using suggested optimum conditions was in agreement with model predicted values although the effect on Apigenin content was more complex. It was further optimized using the help of the model equation.

Molecular, physiological, and biochemical characterization of extracellular lipase production by Aspergillus niger using submerged fermentation.

BackgroundExtracellular production of fungal lipases especially the lipases obtained from the Aspergilli has gained immense interest in recent years due to its diverse biotechnological applications. In this study, we focused on determining the fermentation parameters required for the optimal lipase production.MethodsA total of 256 fungal isolates were obtained from oil seeds. From each genus, one isolate was selected to evaluate lipase production using phenol red and tributyrin plate assays. Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. The highest lipase producer isolates were identified using 18S ribosomal RNA gene sequencing. The genetic variability was determined by random amplified polymorphic DNA (RAPD) analysis and the dendrogram was constructed using the unweighted pair group method with arithmetic averages method. The isolates were examined in a submerged fermentation culture (Smf) to measure the effect of temperature, pH, incubation time, carbon source, nitrogen source, inoculum volume, and lipid source on lipase production.ResultsEleven isolates belonging to the genus Aspergillus were analyzed for lipase production where they were found to be the highest lipase producers among various fungal genera. All the tested isolates were identified as A. niger using 18s rRNA sequencing. Genetic diversity was evaluated among all of the studied A. niger isolates using RAPD primers. The RAPD primers were used to amplify 285 loci, of which five were polymorphic (1.75%) and seven were monomorphic (2.45%). Thus, a high level of genetic diversity was observed among all isolates. The tributyrin test and the lipase activity assay identified five strains of A. niger as high lipase producers, and their optimal enzyme activities were 709.74, 532.54, 735.64, 794.62, and 787.69 U/ml. The optimal conditions for lipase production were as follows: 40 °C, pH 7.5, 1% fructose as the carbon source, 1% yeast extract as the nitrogen source, 2% palm oil, 2.5 × 107 spores/ml suspension, and 3 days of incubation.ConclusionsThe current study provides a comprehensive characterization of the optimal conditions, which are essential to enhance lipase production in five A. niger isolates.

Optimization of the fermentation conditions of Bacillus nematodes and screening of macroporous adsorption resin

Bacillus sp. SMrs28, metabolites from which had significant nematicidal activity, was isolated from the rhizosphere soil of Stellera chamaejasme. To determine the optimal fermentation conditions of the strain and the resin type of preliminary purified active ingredient, fermentation conditions were optimized by single factor experiment, while the macroporous resin types were screened in a static adsorption experiment. The results showed that the optimal fermentation conditions of SMrs28 strain were as follows: glucose and yeast powder were the best carbon source and nitrogen source, fermentation for 48 h, inoculum volume of 10%, temperature at 28 ℃,a rotation speed of 180 r·min-1, liquid volume of 30 mL in 150 mL triangular flask, and with an initial pH of 7.2. The static adsorption experiments showed that the adsorption and desorption of active ingre-dients in the fermentation broth by the macroporous adsorption resin D101 was significantly better than that of XAD-4, HP20 and AB-8, with the nematicidal activity of the desorption liquid being significantly improved. The nematicidal activity of fermentation broth was significantly improved by the optimization of fermentation conditions and the screening of optimal macroporous adsorption resins. These results laid a foundation for the further isolation and purification of active ingredients from SMrs28 strain, and provided theoretical basis for the development and utilization of microbial nematicides.

Establishment of regenerative callus, cell suspension system, and molecular characterization of Taxus wallichiana Zucc. for the in vitro production of Taxol

Taxus wallichiana, an endangered Taxus species native to Nepal, is a source of Taxol (paclitaxel), an anticancer drug, which if produced via plant cell culture in vitro not only reduces the pressure on natural habitat but also allows continuous production throughout the seasons. Gamborg’s B5 medium was the culture medium to develop callus and suspension culture. Quantification of Taxol was carried out via high performance liquid chromatography (HPLC) using C18 column, water-acetonitrile mobile phase, and 1 ml/minute flowrate at 227 nm wavelength. Genetic variation among population was determined using the POPGEN32, version 1.31. Best callus response was in medium with 1 mg/l 2,4-D for both stem and needle explants. For cell suspension culture, callus from needle explants showed better response compared to stem. Fructose and sucrose proved to be the best carbon sources. Cell count increased as carbon concentration increased. 2,4-D at low concentration (5 mg/l) showed higher cell growth rate. Inoculum volume was directly proportional to cell count. Extra addition of macronutrients had inhibitory effect on cell count, but the effect was reversed for MgSO4. Using HPLC, for bark, the highest Taxol yield was 0.057 mg/g distilled water (DW), while for needle was 0.056 mg/g DW. For suspension cultured HPLC samples, Taxol yield ranged at 0.004%. Size range of amplified Deoxyribonucleic acids was between 200 and 2,000 bp. Thus, this investigation generated a concrete data for Taxus population of Nepal providing efficient protocols for establishment of callus and cell suspension culture with the aim of producing Taxol in in vitro conditions.

Partial consolidated bioprocessing of pretreated Pennisetum sp. by anaerobic thermophiles for enhanced bioethanol production

Rapid industrialization and consumption of fossil fuels have led to considerable progress in the production of renewable biofuels like bioethanol. Lignocellulosic biomass such as grasses serves as cheap feedstocks for the production of bioethanol. However, the process involved in lignocellulosic bioethanol production is expensive which restricts its industrial production. The present study thus attempted to investigate a partially consolidated bioprocessing (PCB) approach using two isolated anaerobic thermophiles i.e. Bacillus paranthracis and Bacillus nitratireducens for direct conversion of ultra-sonication assisted sodium hydroxide (UA-NaOH) pretreated Denannath grass to bioethanol in co-culture consortium batch fermentation experiments. The process parameters for the PCB approach were optimized using the Box-Behnken design of Response Surface Methodology (RSM). The parameters that were considered were substrate concentration (5–10 g), incubation time (30–66 h), inoculum volume [1:1 to 3:3 (% v/v) and temperature (50–65 °C). The maximum ethanol concentration of 8.46 mM (0.39 g/L from 7.5 g/L of substrate loading) and ethanol yield (Yp/s) of 0.55 g/g of reducing sugar was obtained at 57.5 °C. In the same conditions the cellulase and xylanase activities were 0.8 U/mL and 11.53 U/mL respectively, while the lactate and acetate concentrations were 0.2 mM (0.009 g/L) and 2.9 mM (0.13 g/L) correspondingly. An increase in the substrate loadings to 250 g/L in a batch fermenter (3 L) resulted in the production of 373.35 mM (17.1 g/L) of ethanol concentration and Yp/s of 0.16 g/g of reducing sugar.

Biochemical and kinetic evaluation of lipase and biosurfactant assisted ex novo synthesis of microbial oil for biodiesel production by Yarrowia lipolytica utilizing chicken tallow

The present study explores the production of biodiesel, a sustainable replacement for depleting fossil fuel by utilizing microbial oil, which was procured from Yarrowia lipolytica employing chicken tallow as the carbon substrate. Chicken tallow, yeast extract, and MgSO4·7H2O were screened for biomass production through Plackett–Burman design. Further, Box–Behnken design analysis was performed, and the optimal concentration of the medium variables was found to be 20 g/L of chicken tallow, 7.0 g/L of yeast extract, and 0.45 g/L of MgSO4·7H2O.The various parameters viz., pH (6), temperature (30 °C), RPM (150), inoculum volume (5%, v/v), and C/N ratio (100) were optimized for maximal biomass and lipid yield, and lipid content. Nile red-stained cells were observed for intracellular lipid bodies using fluorescence microscopy, and its fluorescence intensity was measured bythe flow cytometer. The dimorphic transition and substrate assimilation of Y. lipolytica were analyzed using scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Batch kinetic studies revealed the concomitant synthesis of microbial lipid (4.16 g/L), lipase (43 U/mL), and biosurfactant (1.41 g/L). The GC-MS analysis of microbial oil presented the fatty acid profile as oleic acid (49.15%), palmitic acid (29.83%), stearic acid (11.43%), linoleic acid (3.83%), palmitoleic acid (3.77%), and myristic acid (1.32%).