Isolation, Characterization and Growth Optimization of Glufosunate Ammonium Degrading Bacteria from Farmlands Soil in Mubi

Lipases are triacylglycerol hydrolaseswith various potential applications because of their different physical properties. Most lipase producers are extracellular in nature and are created using solid-state fermentation and submerged fermentation methods. The fungal, mycelial, and yeast lipases are produced using various solid substrates through the solid-state fermentation method. This method is cost-effective and widely used in industries to produce lipase using fungi. However, lipases from bacteria are produced using submerged fermentation. The optimization of media is a main requirement for increasing the quantitative yield by the overproduction of enzymes. The optimization of media is a main requirement for increasing the quantitative yield by overproduction of enzymes. Different parameters, such as pH, temperature, agitation speed, inoculum size, incubation time, and carbon and nitrogen sources, have been of great importance for researchers in designing economical media. The optimization by one factor at a time (OFAT) is a one-dimensional approach that is laborious and time-consuming and does not consider interactions between the factors. The limitations of OFAT method can be alleviated by employing some techniques, such as Plackett-Burman design (PBD) and response surface methodology (RSM). The PBD is a method to screen the variables that influence production and remove the non-significant factors to attain a smaller and manageable set of factors. Subsequently, the chosen significant factors are optimized by RSM that assists to study the interactions of different factors. The RSM comprises of central composite design (CCD) to fit a second-order polynomial equation. In this study, the effect of temperature, tryptone, inoculum size, and incubation time on the lipase production were analysed by PBD screening experiments. The experiments were designed using a CCD with four variables as part of RSM, utilizing the Design Expert software. This model predicted optimal activity of lipase at 58.53 U/mL when using 1.5% tryptone, a 10 mL inoculum size, and an incubation period of 48 h at 34°C. This experiment was further validated and optimal activity of lipase of 57.85 U/mL was observed. Thus, RSM model enhanced the production of lipase and can be applied for the maximum yield of lipase.

Lipase enzymes possess a wide range of industrial applications. Thus, the capacity of lipase-producing bacteria to proliferate on tributyrin agar medium was used to screen for them among various sources. The lipase producing bacteria with highest zone of clearance on the screening media were preserved on the agar slants. The preserved slants were characterized by16SrRNA gene sequencing. The nucleotide sequence so obtained by the 16SrRNA gene sequence was then put through phylogenetic analysis and homology search using the NCBI’s BLAST program. The sequence of lipase producing bacteria showed maximum resemblance with Enterobacter hormaechei bacterial strain. These bacterial strains were produced by inoculating the culture in the inoculum media and allowing it to enrich over night. Subsequently, 3% of the inoculum from the inoculum media was added to the production media, which was then incubated for 48 hours in a rotary shaker. After production the media was centrifuged and supernatant was extracted and used further for optimization, Optimization of the physiochemical parameters of the bacterial strain like inoculum pH, incubation period, inoculum size was found using one factor at a time (OFAT) approach and medium parameters like different carbon source, nitrogen source, substrate, minerals, salts (11 factors) were screened using Plackett-Burman (PB) design which is a full factorial design. The Lipase activity was found by using a titrimetric method using olive oil and Arabic gum mixture as substrate mixture. The maximum lipase activity was found for inoculum pH of 5, 48 hours of incubation time, and 5% inoculum size. The results of the PB design showed the significant parameters to be glucose (carbon source), peptone (nitrogen source), KH2PO4 (salt), and NaCl (mineral). The organism of the soil sample containing bacterial strain showed maximum lipase activity of 70 U/ml and protein concentration of 4.3 μg/mL at the optimized conditions. After centrifuging the culture media that had been improved, the supernatant was collected and partially purified using dialysis and the ammonium sulfate precipitation procedure. Following precipitation, the supernatant’s activity was measured to be 74 U/mL. After being gathered, the pallet was dialyzed in a dialysis bag and added to a buffer. Both the protein content and the lipase activity were estimated. The protein concentration determined by Lowry’s technique was found to be 6.2 μg/mL, and the lipase activity was reported to be 85.22 U/mL.

Bioremediation of lead contaminated environment by Bacillus cereus strain BUK_BCH_BTE2: Isolation and characterization of the bacterium

Optimization of process parameter for alpha-amylase produced by Bacillus cereus amy3 using one factor at a time (OFAT) and central composite rotatable (CCRD) design based response surface methodology (RSM)

Penicillium sp. JUFP2, a strain previously isolated from the soil collected from the Agumbe Forest of the Western Ghats, had potential cytotoxicity against Human Cervical Cancer Cell Line HeLa. It was found to be an uncharacterized, potential candidate in the search for novel anticancer drugs, and hence a need to produce bioactive (anticancer) compounds in substantial quantities. One method of accomplishing this is through the optimization of culture conditions including, carbon and nitrogen sources, incubation period, pH and temperature. A combination of techniques comprising of classical one factor at a time (OFAT) approach, statistical Taguchi and Response surface methodology (RSM) approach were used. OFAT technique was used to select the ideal carbon and nitrogen sources, incubation period, pH and temperature from a panel, Taguchi design was used to study the effect of interaction of different factors on enhancement and RSM was used to determine the optimum concentration of each of the factors. Ammonium sulphate, urea and sodium nitrate were found as suitable factors which enhanced both biomass and metabolite production by Taguchi method. From RSM, ammonium sulphate at 0.08 % (w/v), urea at 0.05 % (w/v) and sodium nitrate at 0.05 % (w/v) in the medium with incubation period of 10 days at a pH of 5.5 with Czapek Dox Yeast broth as basal medium was found to produce the highest quantity of biomass and metabolite from Penicillium sp. JUFP2. The bioactive fraction production was found to be significantly enhanced from un-optimized to optimized conditions.

Cantharellus cibarius is a wild edible mushrooms and considered as a plethora of compounds with potential biotechnological applications. This study highlighted the utilization of C. cibarius mushroom in the production of extracellular lipase under submerged fermentation, representing the first report on lipase production by this mushroom. Various physicochemical factors were optimized via one-factor-at-a time (OFAT) method. Maximum enzyme production was recorded when the mushroom mycelium was grown at 30°C on pH 6.0 for 96h in the medium supplemented with 1% [(v/v)] olive oil. Productivity of enzyme was affected by variation in the nitrogen sources, carbon sources, metal ions and NaCl salt. Glucose and peptone significantly enhanced enzyme production as carbon and nitrogen sources, respectively. Stimulatory and inhibitory effects were found by Ca2+ and Zn2+ ions, respectively. Furthermore, Box-Behnken Design (BBD) of Response Surface Methodology (RSM) was employed to optimize the interactive effects of specific media components like glucose, olive oil and CaCl2. The regression model was significant with a coefficient of determination (R2) value of 0.9483. Statistically optimized design (RSM) resulted approximately two-fold increase (23.5-42.283UmL-1) of lipase production than classical optimization method (OFAT), confirmed the validation of model. The kinetic parameters for p-nitrophenyl palmitate hydrolysis, Kmand Vmax were 5.24mM and 0.768mmol/min/mg respectively, established a high affinity for the substrate.

This article delves into the application of design of experiments methodologies for the integration of a second-order definitive screening design (DSD) and artificial neural network (ANN) to comprehensively assess and predict nine operational variables aimed at increasing the yield of α-amylase from Bacillus velezensis species. By utilizing environmentally friendly and cost-effective agro-solid substrates such as moong husk and soya bean cake, the physical and chemical parameters influencing α-amylase biosynthesis from B. velezensis species were optimized, gathering early data from experiments conducted in shake flasks through the standard one-factor-at-a-time (OFAT) technique. In the realm of response surface methodology (RSM) utilizing the DSD model, nine process variables were taken into account, including pH, temperature, carbon source, nitrogen source, K2PO4, MgSO4, NaCl, fructose, and NaNO3. Furthermore, optimization based on ANN modeling was employed to enhance the enzyme yield further. Experiments were then executed under the optimal conditions as defined by RSM and ANN to corroborate the predicted optimized enzyme activity. As a result, B. velezensis species exhibited enzyme activity of 1092.49U/mL under the optimal process variables identified by both RSM and ANN optimization methods, which included pH 5.48, temperature (34.28°C), carbon source (4.09%), nitrogen source (2.02%), K2PO4 (0.34%), MgSO4 (0.14%), NaCl (0.23%), fructose (1.54%), and NaNO3 (0.53%). To encapsulate, compared to the OFAT technique, where the enzyme activity was 418.25U/mL, a 2.6-fold increase in enzyme activity was achieved by integrating DSD and ANN optimization, considering only nine significant process parameters for the proliferation of B. velezensis species and the maximization of α-amylase activity. The α-amylase enzyme from the B. velezensis species was further purified and characterized. The purification process achieved a 71.77-fold increase in specific activity, with the purified enzyme exhibiting optimal activity at pH 5.5 and 55°C. The enzyme displayed high thermal stability, with minimal activity loss up to 4°C. Kinetic analysis revealed a KM of 0.85mg/mL and a Vmax of 250U/mg/min. The enzyme was found to be metal-independent, with inhibition observed for certain metal ions.

Cellulases are considered to be among the most important enzymes in the commercial market and in various industries. Their applications are widespread, leading to increased demand and high associated costs. This necessitates the search for more cost-effective cellulases from microorganisms. Therefore, the aim of this study was to optimize cellulase production by Trichoderma harzianum PK5 using the One Factor at a Time (OFAT) approach. The effects of carbon, nitrogen, and various environmental factors were studied in both submerged and solid-state fermentation setups by adjusting one factor at a time based on the optimal conditions established from the previous condition. Copra meal and KNO3 were identified as the best complex carbon and nitrogen sources, respectively, for cellulase production by Trichoderma harzianum PK5. The optimal pH of 4.0, moisture concentration of 125% (v/w), inoculum size of 8%, temperature of 30°C, and an incubation time of 7 days were determined as the optimal conditions for cellulase production by this isolate, resulting in an enzyme titre of 252.54±7.73 U/gds in solid-state fermentation. It was found that cellulase enzyme production by the isolate was constitutive. In conclusion, cellulase production by T. harzianum PK5 was significantly optimized using the OFAT approach.

Glucose oxidase catalyzes glucose into gluconolactone and hydrogen peroxide (H 2 O 2 ). The ability of Aspergillus niger InaCC F539 to produce this enzyme needs to be improved. In this research, glucose oxidase production was optimized through fermentation by A. niger InaCC F539 using pineapple peel waste extract under different factors. First, by optimizing carbon and nitrogen sources using One Factor At a Time (OFAT), then optimizing temperature and pH using the Response Surface Methodology (RSM). Enzyme activity was determined and served as a response variable. The results showed that the highest crude extract activity of glucose oxidase enzyme was obtained at 96 hours of incubation with an activity of 1.15 U/mL with glucose as a carbon source and 1.34 U/mL with peptone as a nitrogen source. RSM revealed optimal conditions at 34 °C and pH 6.3, yielding 0.70 U/mL activity. The crude extract of this enzyme also showed antimicrobial activity against Aggregatibacter actinomycetemcomitans , bacteria that cause periodontitis. This study contributes to increasing the production of glucose oxidase enzymes by utilizing agro-industrial waste and can be recommended in industrial applications.

A bacterial strain, Lactobacillus delbrueckii, was isolated and screened for high potential of biosurfactant (BS) production yield. The current research emphasizes the optimization of process parameters through OFAT (one factor at a time) and RSM (response surface methodology) to achieve optimal BS production. Therefore, xylose and peptone were screened as the best carbon and nitrogen sources respectively by means of OFAT, along with temperature (37°C), inoculum size (5%), and fermentation time (39 h). Furthermore, optimization of xylose (1% w/v), peptone (0.5%w/v), and pH (6.5) through Box–Behnken statistical design was validated by ANOVA (analysis of variance) and found significant (p < .05) with optimal BS yield (7.31 g/L). The solvent‐extracted glycopeptide BS displayed cationic nature, with thermal (0–120°C), pH (2–12), and NaCl (2%–12% w/v) stability. The functional attributes of BS like antiadhesive and antimicrobial activity toward Gram‐positive and Gram‐negative bacteria and emulsifying potency against various vegetable oils make it worthwhile and applicable.Practical applicationsThe recent applications of BS and hazards of pathogenic biofilm make it challenging to produce enhanced yield of BS. In light of this, present work is focused to optimize yield from Lactobacillus delbrueckii for its applications as antibiofilm and bioemulsifier agent in various industries. Furthermore, the glycoproteinaceous BS from microbe in question is itself unique and novel. Additionally, the statistical design of optimization provides experiment modeling, production rate, and significance of opted design which makes the whole process feasible to explore new possibilities for BS production.

Indole 3-acetic acid (IAA) is the principal hormone which regulates various developmental and physiological processes in plants. IAA production is considered as a key trait for supporting plant growth. Hence, in this study, production of indole-3-acetic acid (IAA) by a basidiomycetous red yeast Rhodosporidium paludigenum DMKU-RP301 (AB920314) was investigated and improved by the optimization of the culture medium and culture conditions using one factor at a time (OFAT) and response surface methodology (RSM). The study considered the effects of incubation time, carbon and nitrogen sources, growth factor, tryptophan, temperature, shaking speed, NaCl and pH, on the production of IAA. The results showed that all the factors studied, except NaCl, affected IAA production by R. paludigenum DMKU-RP301. Maximum IAA production of 1,623.9 mg/l was obtained as a result of the studies using RSM. The optimal medium and growth conditions observed in this study resulted in an increase of IAA production by a factor of up to 5.0 compared to the unoptimized condition, i.e. when yeast extract peptone dextrose (YPD) broth supplemented with 0.1% l-tryptophan was used as the production medium. The production of IAA was then scaled up in a 2-l stirred tank fermenter, and the maximum IAA of 1,627.1 mg/l was obtained. This experiment indicated that the obtained optimal medium and condition (pH and temperature) from shaking flask production can be used for the production of IAA in a larger size production. In addition, the present research is the first to report on the optimization of IAA production by the yeast Rhodosporidium.

Xylanase production from an alkalophilic actinomycete isolate Streptomyces sp. RCK-2010, its characterization and application in saccharification of second generation biomass

Alkaline cellulase producing Fusarium oxysporum VSTPDK was isolated and screened from the soil of Kapurthala district Punjab, India. This organism produced endoglucanase (CMCase) and exoglucanase (FPase) when grown at a different range of parameters pH (6, 7, 8, 9, 10 and 11), temperature (30, 40, 50 and 60OC), incubation time (4th 6th 8th 10th and 12th day) and nitrogen source (NH4SO4, NH4Cl, NaNO3 and NH4HCO3). Carboxymethylcellulose (CMC) and cellulose powder were used as the sole carbon source. In this research, statistical tools called Response Surface Methodology (RSM) was used for the optimization of cellulase enzyme by selecting three important parameters after one factor at a time (OFAT) approach. Using OFAT, optimum production of both CMCsase (3.52U/mL) and FPase (4.07U/mL) were achieved after 8 days incubation at pH 8, temperature 30OC and 1.0g/L ammonium sulphate while RSM produced CMCase 3.91U/mL and FPase 4.26U/mL respectively when incubated for 8 days at pH 8.5, temperature 45 and 3% ammonium sulphate concentration. Optimization of the culture conditions using RSM leads to an increase of 0.39U/mL (CMCase) and 0.19U/mL (FPase). The use of RSM has gained considerable attention in the past decade in the optimization of various physicochemical parameters and nutritional factors. Its application in different industries may find ways of selecting different factors influencing cellulase activity. The fungus can produce a considerable amount of cellulase enzyme at a pH of up to 10 and 50OC. To our knowledge, this is the first report of alkalothermophilic oxysporum VSTPDK from Punjab, India.

Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation

The herbicide glyphosate is often used to control weeds in agricultural lands. However, despite its ability to effectively kill weeds at low cost, health problems are still reported due to its toxicity level. The removal of glyphosate from the environment is usually done by microbiological process since chemical process of degradation is ineffective due to the presence of highly stable bonds. Therefore, finding glyphosate-degrading microorganisms in the soil of interest is crucial to remediate this glyphosate. Burkholderia vietnamiensis strain AQ5-12 was found to have glyphosate-degrading ability. Optimisation of biodegradation condition was carried out utilising one factor at a time (OFAT) and response surface methodology (RSM). Five parameters including carbon and nitrogen source, pH, temperature and glyphosate concentration were optimised. Based on OFAT result, glyphosate degradation was observed to be optimum at fructose concentration of 6, 0.5g/L ammonia sulphate, pH 6.5, temperature of 32°C and glyphosate concentration at 100ppm. Meanwhile, RSM resulted in a better degradation with 92.32% of 100ppm glyphosate compared to OFAT. The bacterium was seen to tolerate up to 500ppm glyphosate while increasing concentration results in reduced degradation and bacterial growth rate.