Inoculum Age Research Articles

Optimization of Process Parameters for Production of Alkaline Protease by OVAT Method Using Isolated Strain Alternaria alternata TUSGF1

Aim: The current study aimed at studying the optimum fermentation conditions and nutritional conditions for alkaline protease production by submerge fermentation using Alternaria alternata TUSGF1, isolated from poultry farm soil.
 Study Design: The results of environmental and nutritional parameters for protease production by OVAT method was analyzed by origin 6.1 software.
 Place and Duration of Study: Department of Food technology and Biochemical Engineering, Jadavpur University, Kolkata, West Bengal, India between March 2017 and May 2017.
 Methodology: A protease producing microorganism was isolated from a poultry farm soil and identified as Alternaria alternata TUSGF1. Various environmental and nutritional process parameters such as volume of medium, fermentation time, temperature, age of inoculums, agitation and carbon sources and nitrogen sources were standardized for the maximum yield of alkaline protease.
 Results: The optimum conditions of protease activity was 30°C at volume of medium 60 ml with 7 days age of inoculum in the medium containing 168 h of incubation and 120 rpm agitation rate. Peptone, casien, skimmed milk, urea and yeast extract were good nitrogen sources whilst maltose, fructose, starch, and sucrose were appropriate for enzyme production by submerge fermentation.
 Conclusion: Alkaline protease production by a newly isolated Alternaria alternata TUSGF1 from poultry farm soil was studied in shake flask conditions by submerge fermentation. It was established that the optimum protease production was recorded at 30°C, 60 ml volume of medium leaves and incubation time of 168 h. The best carbon and nitrogen sources for protease production were fructose and casein, respectively.

Optimization of Process Parameters for Cholesterol Oxidase Production by Streptomyces Olivaceus MTCC 6820

Background:Streptomyces olivaceusMTCC 6820 is a potent microorganism for cholesterol oxidase (ChOx) production through the submerged fermentation process. Statistical optimization of the process parameters for submerged fermentation enhances the production of enzymes.Objective:This work is aimed to optimize the culture conditions for the fermentative production of cholesterol oxidase byStreptomyces olivaceusMTCC 6820 using combined Response Surface Methodology (RSM) and Artificial Neural Network (ANN) techniques.Methods:The ChOx production (U/ml) was modeled and optimized as a function of six independent variables (culture conditions) using RSM and ANN.Results:ChOx production enhanced 2.2 fold,i.e1.9 ± 0.21 U/ml under unoptimized conditions to 4.2 ± 0.51 U/ml after the optimization of culture conditions. Higher coefficient of determination (R2= 97.09 %) for RSM and lower values of MSE (0.039) and MAPE (3.46 %) for ANN proved the adequacy of both the models. The optimized culture conditions predicted by RSMvs. ANN were pH (7.5), inoculum age (48 h), inoculum size (11.25 % v/v), fermentation period (72 h), incubation temperature (30°C) and shaking speed (175 rpm).Conclusion:The modeling, optimization and prediction abilities of both RSM and ANN methodologies were compared. The values of Pearson correlation coefficient (r) (ANN0.98> RSM0.95), regression coefficient (R2) between experimental activity, RSM and ANN predicted ChOx activity, respectively (ANN0.96> RSM0.90) and Absolute Average Deviation (AAD) for (ANN3.46%< RSM9.87%) substantiated better prediction ability of ANN than RSM. These statistical values indicated the superiority of ANN in capturing the non-linear behavior of the system.

Optimization of parameters for lipase production by Aspergillus niger NRRL-599 using response surface methodology

Background and objective Lipases are characterized to catalyze precise chemical transformation. That is they have widespread applications in detergents, cosmetics, food, organics synthesis, and pharmaceutical industries. In recent years, the study and development of lipase production in solid state fermentation, also using sequential statistical strategy as optimization for some vital factors are gaining more attention. This may be attributed to several advantages using techniques such as simplicity of fermentation media, no need for complex machinery. Equipment and control system, compactness of fermentation vessel due to lower water volume, high yields, less energy needed, and lower capital. Materials and methods Aspergillus niger NRRL-599 strain was tested for lipase production using different agroindustrial wastes including wheat bran, wheat germ cake oil, jojoba cake oil, almond cake oil, and olive oil as substrates under solid state fermentation. For further optimization, lipase activity was studied using two sequential optimization. Results and conclusion Olive oil waste was the most suitable substrate for lipase production (125 U/ml). Using one variable at a time, maximum lipase activity (200 U/ml) and specific enzyme activity (357.1 U/mg) were recorded in the presence of 5% w/v olive oil, 48 h inoculum age, 4% v/v inoculum size after 14 days fermentation at 30°C. The screening of the seven physiological factors using Plackett–Burman design showed that only three variables; that is tween 80, moisture content, and inoculum age affected significantly lipase production. Optimization by Box–Behnken design resulted in the highest lipase activity (420 U/ml) in which the most effective variables were inoculum size, inoculum age, moisture content, and tween 80 1%.

Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides

An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ∼75 kDa with optimum activity at pH 7.0 and 20 °C. FOS production after parametric optimization (sucrose − 50% w/v, m-FTase dose − 4.5% w/v, inoculum age − 48 h and incubation time − 24 h) reached 325 g/L (55% yield) with 14% residual sucrose, 25% glucose and 6% fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.

Pineapple Waste and Wastewater: Route for Biodiesel Production from Candida tropicalis (MF510172)

Abstract: The aim of the present study was to utilize waste resources for biodiesel production and to test its recyclability. The oleaginous yeast was isolated from local municipal wastewater outlet and on the basis of 18S r RNA sequencing, isolate was identified as Candida tropicalis (MF510172). Optimum fermentation conditions for lipid yield was recorded to be at 180 rpm agitation speed, 6% pineapple waste as a carbon source, 5% inoculum size,48 h inoculum age at 40°C temperature with 72 h incubation time. Candia was able to produce 13 ml. L-1 lipids using optimum conditions. Extracted lipids were transesterified to form fatty acid methyl esters (FAME) and further characterized by using GC-MS and FTIR spectroscopy. Palmitic acid methyl esters and other supplementary hydrocarbon compounds were identified using GC-MS. FTIR spectrum indicates the characteristics FAME peak at 1740 cm-1 with C= O stretching band of methyl esters. The results clearly demonstrate the waste resources containing filtered wastewater and pineapple waste used in the study are effective to produce quality oil for biodiesel production.

Study on Optimizing Nutrition and Fermentation Conditions for Compound <i>Bacillus spp.</i>

In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L; fermentation temperature is 37℃, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.

Development of strategy for simultaneous enhanced production of alkaline xylanase-pectinase enzymes by a bacterial isolate in short submerged fermentation cycle

The aim of this study is to enhance the production of industrially valuable xylanase and pectinase enzymes in short duration, using agrowaste extracted substrates. Conventional cum statistical multifactor analysis approaches were used in order to evaluate the effect of crude extracted substrates, supplemented for the production of xylanase-pectinase enzymes. Incorporation of crude extracted xylan (1.2 mg/ml of inoculum) and pectin (4.8 mg/ml of inoculum) substrates in inoculum resulted in maximal xylanase (320 ± 15) and pectinase titre (90 ± 8) after 48 h, using 2% wheat bran and 2% citrus peel in production medium with 48 h of fermentation time, with one variable factor at a time approach. The best condition obtained after performing statistical multifactor interaction analysis includes 5.50 mg/ml of pectin in inoculum,1.50 mg/ml of xylan in inoculum, wheat bran 3%, temperature 37.5 °C, time 48 h, 7 mg/ml of pectin in production medium, peptone 1.05%, inoculum size 2% and inoculum age of 20 h, with alkaline xylanase activity of 415.22 ± 18.50 IU/ml and alkaline pectinase activity of 109.10 ± 8.80 IU/ml. Activity of different pectinolytic enzymes per ml was also calculated, with 18.98 IU of exo-polymethylgalacturonase, 0.14 IU of endo-polymethylgalacturonase, 80 IU of exo-polygalacturonase, 0.28 IU of endo-polygalacturonase, 1.42 IU of polymethylgalacturonate lyase, 1.47 IU of polygalacturonate lyase, 0.15 IU of pectin esterase. This is the first report mentioning the utilization of crude extracted xylan and extracted pectin in inoculum to get the increment in the activity of both alkaline xylanase-pectinase enzymes simultaneously under short submerged fermentation cycle.

Fermentative Production of Sophorolipid and Purification by Adsorption Chromatography

AbstractA sophorolipid (glycolipid biosurfactant from Starmerella bombicola) has valuable properties in addition to surface active attributes. This sophorolipid was produced as a secondary metabolite by submerged fermentation using hydrophobic (sunflower oil) and hydrophilic (glucose) substrate. The present work investigates the effect of inoculum age, oil feed and incubation period to increase sophorolipid yield. The highest yield of sophorolipid by sunflower oil was accomplished with an inoculum age of 60 h. Maximum substrate consumption and sophorolipid yield were found near about 200 h. At optimized fermentation conditions, 19.2 g/L yield of sophorolipid was achieved. The lactonic sophorolipid which is the major component of crude sophorolipid was separated and purified by adsorption chromatography. Normal phase and reverse phase resin were compared for adsorptive purification among which reverse phase resin provides better purification with 69 % recovery. Further surfactant and antimicrobial properties of crude sophorolipid were studied.

Stenotrophomonas maltophilia Gd2: A potential and novel isolate for fibrinolytic enzyme production.

The bacterium with an ability to produce extracellular fibrinolytic protease was isolated and identified as Stenotrophomonas maltophilia Gd2 based on ribotyping. The in-vitro fibrinolytic profile of this enzyme depicted 73% of fibrin clot dissolution within 4 h. Fibrinolytic enzyme yield influenced by different physiological (incubation time, temperature, agitation and pH), nutritional (macronutrients such as carbon and nitrogen sources) and biological (inoculums age and inoculums concentration) parameters of fermentation which were optimized based on one-factor-at-a-time (OFAT) approach. The enzyme yield improved from 886 to 1795 FU ml−1 upon OFAT; optimized conditions include temperature – 33 °C, pH – 8.0, incubation time – 36 h, agitation – 150 RPM, 3% v/v inoculums and age of inoculum – 18 h. Further optimization of enzyme production was achieved with implementation of Plackett-Burman media designing where the production levels increased to 3411 FU ml−1 and noticed that peptone, pH, dextrose and K2HPO4 was found to be significant factor. This ms reports the highest fibrinolytic enzyme yield with S. maltophilia to that of literature reports.

Comparison of ionic liquids for efficient pretreatment of brewery spent grain

A newly isolated fungus Aspergillus niger RCKH-3 was employed for cost effective production of a complete cellulase system under solid state fermentation (SSF). Overall evaluation criteria approach (OEC) was used as a novel approach for medium engineering to maximize the cellulase production. Among several factors studied, NH4Cl, inoculum age and temperature contributed maximally towards cellulase production. The highest interactions were observed between inoculum age and beef extract, followed by substrate to moisture ratio and PEG 8000. The statistical model provided optimal enzyme production conditions as: 5 g wheat bran; mineral salt solution (pH 5) and containing (g/L); NH4Cl (0.5), beef extract (2.0) and PEG 8000 (3.0) substrate to moisture ratio of 1:3.5 and 30% (v/w) 18 h old inoculum. Under optimum conditions, cellulase production experienced 2.5, 3.0 and 1.5 fold enhancements in FPase, CMCase and β-glucosidase activities, respectively. The estimated production costs ($ per 1000 units) for FPase, CMCase, β-glucosidase were 0.47, 0.21 and 0.04, respectively. Saccharification of office waste paper using crude cellulase resulted in reducing sugar yield of 534 mg/g dry substrate. Cellulase of newly isolated fungi A. niger RCKH-3 shows potential in biofuel applications based on its comparatively higher saccharification abilities and lower production costs.

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.

Xylitol Production by Candida tropicalis 31949 from Sugarcane Bagasse Hydrolysate

Application of microbial fermentation of xylose to produce xylitol can not only turn waste into useful products and conserve resources, but also protect the environment. Currently, more than 100 kinds of microorganisms are known to ferment xylose, among which Candida tropicalis has strong xylose conversion ability and high tolerance to inhibitors such as furfural, acetate, and phenolics in xylose mother liquor. The present study focused on fermenting xylose hydrolysate from sugarcane bagasse to produce xylitol using C. tropicalis strain 31949. The effects of inoculum age, inoculum quantity, and initial xylose concentration on xylitol fermentation by C. tropicalis 31949 were discussed. The following optimum fermentation conditions were determined: inoculum quantity, 10%; inoculum age, 26 h; and initial xylose concentration, 100 g/L. Under these optimal fermentation conditions, C. tropicalis 31949 could ferment sugarcane bagasse hydrolysate, which was pretreated by vacuum concentration and activated carbon detoxification decoloration, to produce 62.9800 g/L xylitol.

Plackett-Burman design for screening of process components and their effects on production of lactase by newly isolated Bacillus sp. VUVD101 strain from Dairy effluent

In the present investigation, a low-cost fermentation medium was designed to achieve the maximum production of lactase from the Bacillus sp. VUVD101 strain through the screening of different nutritional and physical variables, using the Plackett-Burman design. Fourteen variables of the fermentation process were selected: incubation time, temperature, pH, RPM, DO, inoculum size, inoculum age, MgSO4, l-Cysteine, KH2PO4, CaCl2, K2PHO4, corn steep liquor and lactose. The selected variables were evaluated through statistical analysis, based on their significance, coefficient value and standard effect plot. The results suggested that six variables, namely, corn steep liquor, lactose, MgSO4, temperature, pH and RPM, had influence with high confidence levels, while the remaining eight variables did not show a significant effect on production. The analysis of the variance value R2 (0.96) also showed the model used for prediction to be significant (p less than 0.05). The plot for the standard effect for each component and its traits provided accurate data by which to select well-suited variables for further optimization. In comparison with the basal medium, 68% higher enzyme activity was achieved from the model of the optimized medium, and lactase activity was found to be 18.31 U/ml.

Optimization and Characterisation of Thermo Stable Exopolysaccharide Produced from Bacillus licheniformis WSF-1 Strain

Bacillus licheniformis WSF-1 strain isolated from sugar distillery plant was capable of producing a maximum amount of EPS (1.24 g/mL) and dry biomass (3.25 g/mL) respectively. Certain factors like inoculum age and carbon source was standardised in view of obtaining high EPS yield. In this study, 48-h-old culture was identified as the optimum inoculum age for EPS production. In fermentation, as carbon source plays a major contributing factor that influences the EPS production, optimum carbon source was determined by studying the effect of different carbohydrates such as 12% of glucose, sucrose, lactose, maltose, xylose and fructose on EPS production. The best carbon source was identified as sucrose at a concentration of 25% producing 2.9 g/mL of exopolysaccharide. The polysaccharides were hydrolysed and the monomeric components were determined as fructose and glucose by HPLC, followed by FT-IR analysis. The thermal study revealed that the EPS can withstand high temperature of 219.4 °C and had high glass transition temperature of 150.6 °C. These features indicate the thermo stable nature of the exopolysaccharide which can be used for various industrial applications.

Fermentative production of rhamnolipid and purification by adsorption chromatography

ABSTRACTGlycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24 hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96 hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8 g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.

Sulfur Isotope Fractionation by Sulfate-Reducing Microbes Can Reflect Past Physiology.

Sulfur (S) isotope fractionation by sulfate-reducing microorganisms is a direct manifestation of their respiratory metabolism. This fractionation is apparent in the substrate (sulfate) and waste (sulfide) produced. The sulfate-reducing metabolism responds to variability in the local environment, with the response determined by the underlying genotype, resulting in the expression of an "isotope phenotype". Sulfur isotope phenotypes have been used as a diagnostic tool for the metabolic activity of sulfate-reducing microorganisms in the environment. Our experiments with Desulfovibrio vulgaris Hildenborough (DvH) grown in batch culture suggest that the S isotope phenotype of sulfate respiring microbes may lag environmental changes on time scales that are longer than generational. When inocula from different phases of growth are assayed under the same environmental conditions, we observed that DvH exhibited different net apparent fractionations of up to -9‰. The magnitude of fractionation was weakly correlated with physiological parameters but was strongly correlated to the age of the initial inoculum. The S isotope fractionation observed between sulfate and sulfide showed a positive correlation with respiration rate, contradicting the well-described negative dependence of fractionation on respiration rate. Quantitative modeling of S isotope fractionation shows that either a large increase (≈50×) in the abundance of sulfate adenylyl transferase (Sat) or a smaller increase in sulfate transport proteins (≈2×) is sufficient to account for the change in fractionation associated with past physiology. Temporal transcriptomic studies with DvH imply that expression of sulfate permeases doubles over the transition from early exponential to early stationary phase, lending support to the transport hypothesis proposed here. As it is apparently maintained for multiple generations (≈1-6) of subsequent growth in the assay environment, we suggest that this fractionation effect acts as a sort of isotopic "memory" of a previous physiological and environmental state. Whatever its root cause, this physiological hysteresis effect can explain variations in fractionations observed in many environments. It may also enable new insights into life at energetic limits, especially if its historical footprint extends deeper than generational.

A two-stage process facilitating microbial lipid production from N-acetylglucosamine by Cryptococcus curvatus cultured under non-sterile conditions

N-acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely studied for lipid production by oleaginous species. This study demonstrated that Cryptococcus curvatus had a great capacity to convert GlcNAc into lipid with high yield using a two-stage production process. Optimal inoculum age and inoculation size strongly improved the two-stage lipid production efficiency. More interestingly, this process rendered superior lipid production under non-sterile condition. The acetate liberated from GlcNAc was consumed timely, while the NH4+ released was rarely assimilated. Lipid titre, lipid content and lipid yield reached 9.9 g/L, 56.9% and 0.23 g/g, respectively, which were significantly higher than those from the conventional process where cell growth and lipid accumulation were coupled. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oil, suggesting their potential for biodiesel production. These findings strongly supported the two-stage process as an attractive strategy for better techno-economics of the chitin-to-biodiesel routes.

Cost effective production of complete cellulase system by newly isolated Aspergillus niger RCKH-3 for efficient enzymatic saccharification: Medium engineering by overall evaluation criteria approach (OEC)

A newly isolated fungus Aspergillus niger RCKH-3 was employed for cost effective production of a complete cellulase system under solid state fermentation (SSF). Overall evaluation criteria approach (OEC) was used as a novel approach for medium engineering to maximize the cellulase production. Among several factors studied, NH4Cl, inoculum age and temperature contributed maximally towards cellulase production. The highest interactions were observed between inoculum age and beef extract, followed by substrate to moisture ratio and PEG 8000. The statistical model provided optimal enzyme production conditions as: 5 g wheat bran; mineral salt solution (pH 5) and containing (g/L); NH4Cl (0.5), beef extract (2.0) and PEG 8000 (3.0) substrate to moisture ratio of 1:3.5 and 30% (v/w) 18 h old inoculum. Under optimum conditions, cellulase production experienced 2.5, 3.0 and 1.5 fold enhancements in FPase, CMCase and β-glucosidase activities, respectively. The estimated production costs ($ per 1000 units) for FPase, CMCase, β-glucosidase were 0.47, 0.21 and 0.04, respectively. Saccharification of office waste paper using crude cellulase resulted in reducing sugar yield of 534 mg/g dry substrate. Cellulase of newly isolated fungi A. niger RCKH-3 shows potential in biofuel applications based on its comparatively higher saccharification abilities and lower production costs.

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

ABSTRACTPropionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54 ± 0.023 g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48 hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74 ± 0.06 g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.

Characterization of D-lactic acid, spore-forming bacteria and Terrilactibacillus laevilacticus SK5-6 as potential industrial strains

In this study, we screened and isolated D-lactic acid-producing bacteria from soil and tree barks collected in Thailand. Among the isolates obtained, Terrilactibacillus laevilacticus SK5-6 exhibited good D-lactate production in the primary screening fermentation (99.27 g/L final lactate titer with 0.90 g/g yield, 1.38 g/L⋅h, and 99.00% D-enantiomer equivalent). Terrilactibacillus laevilacticus SK5-6 is a Gram-positive, endospore-forming, homofermentative D-lactate producer that can ferment a wide range of sugars to produce D-lactate. Unlike the typical D-lactate producers, such as catalase-negative Sporolactobacillus sp., T. laevilacticus SK5-6 possesses catalase activity; therefore, a two-phase fermentation was employed for D-lactate production. During an aerobic preculture stage, a high-density cell mass was rapidly obtained due to aerobic respiration. When transferred to the fermentation stage at the correct physiological stage (inoculum age) and proper concentration of cell mass (inoculum size), T. laevilacticus rapidly converted glucose into D-lactate under anaerobic conditions, resulting in a high final lactate titer (102.22 g/L), high yield (0.84 g/g), and high productivity (2.13 g/L⋅h). When the process conditions were shifted from an aerobic to an anaerobic environment, unlike other lactate-producing bacteria, the mixed acid fermentation route was not activated in the culture of T. laevilacticus SK5-6 during the fermentation stage when some trace oxygen still remained. Our study demonstrates the excellent characteristics of this isolate for D-lactate production; in particular, a high product yield was obtained without byproduct formation. Based on these key characteristics of T. laevilacticus SK5-6, we suggest that this isolate is a novel D-lactate producer for use in industrial fermentation.