Computerized study of interactions among factors and their optimization through response surface methodology for the production of tannin acyl hydrolase by Aspergillus niger PKL 104 under solid state fermentation

Optimization of five parameters (initial moisture, initial pH, incubation temperature, inoculum ratio and fermentation period), as per central composite rotable design falling under the response surface methodology, was attempted in a total of 32 experimental sets, after fitting the experimental data to the polynomial model of a suitable degree, for tannin acyl hydrolase production by Aspergillus niger PKL 104 in solid state fermentation system. The quantitative relation between the enzyme production and different levels of these factors was exploited to work out optimized levels of these parameters by flexible polyhedron search method and confirmed by further experimentations. The best set required 5% inoculum, 6.5 initial pH, 28 °C fermentation temperature, 62% initial moisture and 3 days fermentation time. The optima were worked out under the additional constraints for temperature ( 30 °C) and fermentation time (not more than 3 days) which are essential from industrial conditions and to pre-empt contamination, respectively. The best set resulted in 1.34 times more enzyme production than that was obtained before this optimization. Three dimensional plots, relating the enzyme production to paired factors (when other three factors were kept at their optimal levels) best described the behaviour of solid state fermentation system and the interactions between factors under optimized conditions. The model showed that the enzyme production was affected by all the five factors studied. The initial pH exhibited a positive interaction with moisture but no interaction with other factors. Initial moisture level and inoculum ratio showed negative interaction in contrast to positive interaction between inoculum ratio and fermentation period. It is thus apparent that the response surface methodology not only gives valuable information on interactions between the factors but also leads to identification of feasible optimum values of the studied factors, in addition to 99% (or more) savings on resources as compared to a full factorial traditional optimization method. Response surface methods have not been used earlier for optimizing parameters in solid state fermentation system.The authors thank Dr. S. R. Bhowmik, Director, CFTRI for the interest shown in the work. P. K. Lekha is thankful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of a research fellowship.     

Review of solid state fermentation for lignocellulolytic enzyme production: challenges for environmental applications

Within the context of increasing environmental concern, energy production from lignocellulosic substrates is gaining great interest. Enzymes have proven their efficiency in the degradation of the lignocellulosic complex but their use remains limited in environmental applications such as anaerobic digestion mainly due to their prohibitive cost. Therefore, solid state fermentation (SSF) emerges as an interesting alternative for the in situ production of lignocellulolytic enzymes. Various research efforts on the lab scale optimization of SSF are discussed. They are presented according to the type of inoculum used in the process: bacterial species and fungal species under both mesophilic and thermophilic conditions. In general, parameters that impact the SSF process include: substrate type and particle size, substrate pretreatment, inoculum, nutrient supplementation, moisture content, pH, aeration, temperature and mixing. Using different substrates, authors aim at maximizing enzyme production taking into account one to several of the indicated operational parameters. The reviewed research puts forward the adaptation of the operational parameters, enzyme production cost and loading, enzyme mixture quality and efficiency and finally reactor design as the main challenges for environmental large-scale application.     

Use of a Fractional Factorial Design to Evaluate Interactions of Environmental Factors Affecting Biodegradation Rates

For investigation of main and interactive effects of six experimentally controlled environmental factors on phenol biodegradation in a shake-flask system, a largely neglected statistical procedure was applied. A major benefit resulting from the application of the orthogonal, fractional factorial design is that the number of experiments necessary to evaluate multifactor interactions is limited. In our investigation, the required number of experiments was reduced to 81 from the 324 necessary with conventional factorial designs; information was sacrificed for only 3 of 15 possible two-factor interactions. Six experimentally controlled factors were investigated at two or three treatment levels each; the six factors were (1) amount of phenol substrate, (2) amount of bacterial inoculum, (3) filtration of inoculum, (4) type of basal salts medium, (5) initial pH of basal salts medium, and (6) flask closure. Significant main effects were found for factors 1, 2, and 4; whereas significant interactive effects were found only for factor 2 with factor 3 and for factor 2 with factor 5. Our results suggest that the application of these statistical designs will greatly reduce the number of experiments necessary to evaluate multifactor effects on degradation rates during optimization of both hazard screening systems and waste treatment systems.     

OPTIMIZATION OF SOLID-STATE FERMENTATION FOR PHYTASE PRODUCTION BY Thermomyces lanuginosus USING RESPONSE SURFACE METHODOLOGY

A strain of Thermomyces lanuginosus, isolated from hot spring water in Turkey, was studied for optimization of phytase production using solid-state fermentation. Effects on fermentation of different production parameters such as substrate type, moisture, culture time, and inoculum size were investigated using a one-factor-at-a-time approach. Central composite design (CCD) of response surface methodology was applied for the optimization of four factors (culture temperature, initial pH, aeration area, age of seeding culture) that were affecting phytase production by Thermomyces lanuginosus in rice bran. Maximum phytase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55°C, initial pH 7.5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, and ZnSO₄ 2.5 mM. An overall 10.83-fold enhancement in phytase activity (0.30 to 3.248 U) was attained due to the optimization.     

L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization

AIMS: Evaluation of fermentation process parameter interactions for the production of l-asparaginase by isolated Staphylococcus sp. - 6A. METHODS AND RESULTS: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. CONCLUSION: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total l-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: All selected factors showed impact on l-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.     

施氏假单胞菌YC-YH1的萘降解特性及产物分析

【目的】萘是一种重要的环境污染物,它在环境中的积累会对人类健康造成危害,生物降解是解决这一问题的有效方法。本实验室保存的施氏假单胞菌YC-YH1对萘具有较强的降解能力,在此基础上,研究和分析菌株对萘的降解特性、环境因素对萘降解率的影响以及代谢产物。【方法】本文首先采用单因素实验法研究pH、温度、接种量、萘初始浓度对萘降解率的影响;并在单因素实验结果的基础上,利用Design-Expert 8.0.5软件和Box-Behnken设计对pH、温度、接种量3个影响因素进行响应面优化分析,建立环境因素对萘降解率影响的优化模型。利用LC-MS检测萘降解过程中产生的重要代谢产物,从而推测菌株对萘的代谢途径。【结果】响应面分析结果表明,优化模型极显著(P<0.001),拟合度良好,预测结果可信度高。降解实验证明,在培养温度为32.4 °C、pH为7.10、接种量5.74% (体积比)的优化条件下培养3 d即可将浓度为100 mg/L的萘100%降解。LC-MS分析表明,菌株降解萘的过程中,能够被检测到的主要代谢产物有1,2-二羟基萘、水杨酸、邻苯二酚等。【结论】施氏假单胞菌YC-YH1对萘有高的降解效率,pH、温度、接种量3个因素对菌株的降解率有较大影响。利用响应面法优化菌株对萘的降解条件,能够提高YC-YH1菌株对萘的生物降解性能。初步推测菌株YC-YH1对萘的降解是通过水杨酸途径,萘首先被其代谢为1,2-二羟基萘,而后被转化为水杨酸和邻苯二酚,最后进入三羧酸循环被彻底降解。     

Optimization of environmental parameters for biodegradation of alpha and beta endosulfan in soil slurry by Pseudomonas aeruginosa

Aim: To determine optimal environmental conditions for achieving biodegradation of α‐ and β‐endosulfan in soil slurries following inoculation with an endosulfan degrading strain of Pseudomonas aeruginosa. Methods and Results: Parameters that were investigated included soil texture, soil slurry: water ratios, initial inoculum size, pH, incubation temperature, aeration, and the use of exogenous sources of organic and amino acids. The results showed that endosulfan degradation was most effectively achieved at an initial inoculum size of 600 μl (OD = 0·86), incubation temperature of 30°C, in aerated slurries at pH 8, in loam soil. Under these conditions, the bacterium removed more than 85% of spiked α‐ and β‐endosulfan (100 mg l^?1) after 16 days. Abiotic degradation in noninoculated control medium within same incubation period was about 16%. Biodegradation of endosulfan varied in different textured soils, being more rapid in course textured soil than in fine textured soil. Increasing the soil contents in the slurry above 15% resulted in less biodegradation of endosulfan. Exogenous application of organic acids (citric acid and acetic acid) and amino acids (l ‐methionine and l ‐cystein) had stimulatory and inhibitory effects, respectively, on biodegradation of endosulfan. Conclusion: The results of this study demonstrated that biodegradation of endosulfan by Ps. aeruginosa in soil sediments enhanced significantly under optimized environmental conditions. Significance and Impact of the Study: Endosulfan is a commonly used pesticide that can contaminate soil, wetlands and groundwater. Our study demonstrates that bioaugmentation of contaminated soils with an endosulfan degrading bacterium under optimized conditions provides an effective bioremediation strategy.     

木质素降解菌BYL-7的筛选及降解条件优化

【背景】微生物降解木质素因其具有降解效率高和环保等特点而备受关注。【目的】筛选高效木质素降解真菌,并对其降解条件进行优化。【方法】通过愈创木酚-马铃薯葡萄糖琼脂(potato dextrose agar,PDA)和苯胺蓝平板法筛选高效木质素降解菌株,利用单因素筛选及响应面试验对培养条件进行优化。【结果】筛选到一株高效木质素降解菌BYL-7,经形态和多序列分析初步确定为Trametes versicolor。单因素试验证明初始pH、温度和接种量为降解木质素显著影响因子,响应面试验确定降解木质素最优条件为：初始pH 6.7,温度25 °C,接种量8%。在此条件下,碱性木质素降解率为36.5%,比未优化前提高54.0%;水稻秸秆木质素、半纤维素和纤维素降解率分别为32.8%、21.5%、13.2%,其中木质素降解率比未优化前提高36.1%;漆酶活性在第6天达到峰值120.0 U/L,比未优化前提高25.0%;木质素过氧化物酶活性在第6天达到峰值1 343.8 U/L,比未优化前提高36.0%;锰过氧化物酶活性在第5天达到峰值463.8 U/L,比未优化前提高31.7%。【结论】研究结果为木质素的降解提供了良好的菌种资源,同时也为后续木质素的研究积累了相关数据。     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.     

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

Isolation and characterization of distillery spent wash color reducing bacteria and process optimization by Taguchi approach

The ability of five different types of bacterial strains isolated from a distillery mill site was analyzed for decolorization of distillery spent wash. 16S rDNA based denaturing gradient gel electrophoresis (DGGE) and amplified random DNA restriction analysis (ARDRA) were used to characterize the bacterial strains. One of the isolates had higher capability to reduce color (21%) and chemical oxygen demand (COD) (30%) was finally identified by 16S rDNA sequence analysis as Bacillus sp. Different parameters such as pH, temperature, aeration,% carbon,% nitrogen, inoculum size and incubation time were optimized by the Taguchi approach to achieve maximum decolorization of distillery spent wash by the Bacillus sp. Reduction in color (85%) and COD (90%) was observed within 12 h after optimization by the Taguchi method. The significant factor in the optimization process was duration followed by inoculum size to attain maximum color reduction. The Taguchi approach proved to be a reliable tool in optimizing culture conditions and analyzing interaction effects of process parameters in achieving the best possible combination for maximum decolorization of the distillery spent wash.     

One-step process for debromination and aerobic mineralization of tetrabromobisphenol-A by a novel Ochrobactrum sp. T isolated from an e-waste recycling site

A novel bacterium, Ochrobactrum sp. T, capable of simultaneous debromination and aerobic mineralization of tetrabromobisphenol-A (TBBPA), was isolated from a sludge sample collected from an electronic-waste recycling site. The bacterium exhibited maximal debrominase activity at pH 6.5, 35 °C, and 200 rpm in Luria-Bertani culture medium. Initial TBBPA concentration and pH had more significant effects on degradation efficiency than those of temperature and inoculum size. Degradation and debromination efficiencies of 91.8% and 86.7%, respectively, were achieved within 72 h under optimized conditions of 35 °C, pH 7.0, inoculum volume of 25 mL, and TBBPA concentration of 3 mg L⁻¹. In addition, a 35.6% decrease in total organic carbon was observed after the degradation of 5 mg L⁻¹ TBBPA for 120 h. Eight metabolic intermediates were identified during the biodegradation of TBBPA. This study is the first report to propose a one-step process for TBBPA debromination and mineralization by a single bacterial strain.     

Poly(L-lactide)-degrading enzyme production by Actinomadura keratinilytica T16-1 in 3 L airlift bioreactor and its degradation ability for biological recycle

The optimal physical factors affecting enzyme production in an airlift fermenter have not been studied so far. Therefore, the physical parameters such as aeration rate, pH, and temperature affecting PLA-degrading enzyme production by Actinomadura keratinilytica strain T16-1 in a 3 l airlift fermenter were investigated. The response surface methodology (RSM) was used to optimize PLA-degrading enzyme production by implementing the central composite design. The optimal conditions for higher production of PLA-degrading enzyme were aeration rate of 0.43 vvm, pH of 6.85, and temperature at 46° C. Under these conditions, the model predicted a PLA-degrading activity of 254 U/ml. Verification of the optimization showed that PLA-degrading enzyme production of 257 U/ml was observed after 3 days cultivation under the optimal conditions in a 3 l airlift fermenter. The production under the optimized condition in the airlift fermenter was higher than un-optimized condition by 1.7 folds and 12 folds with un-optimized medium or condition in shake flasks. This is the first report on the optimization of environmental conditions for improvement of PLA-degrading enzyme production in a 3 l airlift fermenter by using a statistical analysis method. Moreover, the crude PLA-degrading enzyme could be adsorbed to the substrate and degraded PLA powder to produce lactic acid as degradation products. Therefore, this incident indicates that PLA-degrading enzyme produced by Actinomadura keratinilytica NBRC 104111 strain T16-1 has a potential to degrade PLA to lactic acid as a monomer and can be used for the recycle of PLA polymer.     

Corn husk as a novel substrate for the production of rifamycin B by isolated Amycolatopsis sp. RSP 3 under SSF

Rifamycin B production by isolated Amycolatopsis sp. RSP 3 was investigated under solid state fermentation (SSF) using agro-industrial waste materials. Corn husk was the most suitable substrate/support material with 4-fold higher production than wheat bran and corn cobs. A two-level (conventional and statistical) methodology was used to optimize fermentation parameters belong to physiological (pH, temperature and aeration), nutritional (carbon and nitrogen sources) and microbial (inoculum level and incubation time). Conventional optimization significantly improved (450%) the rifamycin B production of which two-third was associated with carbon and nitrogen sources. Starch as carbon source showed negative impact. Statistical optimization of suggested potassium nitrate (at individual level), soya bean meal and barbital (at interactive level) were observed to be the most noticeable variables in the maximization of production. At optimized conditions, inorganic nitrogen source played vital role (>59%) compared to all other factors. Overall, more than 920% increase in rifamycin B production was achieved at optimized environment.     

β - glucosidase production by Aspergillus wentii in stirred tank bioreactors

Summary Batch cultivation of Aspergillus wentii, Wehmer, Pt 2804 was carried out on Mandels and Reese (1957) medium containing 3% cellulose and 0.3% peptone at 30°C in 14 and 30 liter Bioreactors. Use of a 48 hours old inoculum (10% v/v), 0.5 vvm aeration at 400–800 rpm and maintenance of dissolved oxygen and pH above 50% saturation level and between 5.2 to 4.0 respectively, produced 10.58 g cells/lit and 61.9 IU/1/hr cellobiase productivity.     

Changes in interactions between juniper and mistletoe mediated by shared avian frugivores: parasitism to potential mutualism

Although mistletoe is typically viewed as a parasite of juniper in a two-way interaction, its role may become neutral or even mutualistic when their common avian seed dispersing agents are considered as a three-way interaction. In the study area, wintering avian frugivores forage on both one-seed juniper (Juniperus monosperma) berries and on the fruit of its associated mistletoe (Phoradendron juniperinum). Three major findings emerged from our studies that support a three-way interaction and the hypothesis of conditional interactions. First, mistletoes provide a stable resource for shared avian seed dispersers; junipers do not. Whereas juniper berry production varied 10- to 15-fold over the 3 years of our study, mistletoe fruit abundance did not vary significantly. Second, the abundance of avian seed dispersal agents, such as Townsend's solitaires (Myadestes townsendi), is strongly tied to the abundance of juniper berries in mast years and mistletoe fruits in all years. In fact, the best overall predictor of their common avian seed dispersal agents was the abundance of mistletoe; stands with mistletoe attracted up to 3 times more avian frugivores than stands with little or no mistletoe. Thus, mistletoe berries can serve as the main attractor for birds that disperse juniper berries. Third, in agreement with the hypothesis that mistletoe can benefit junipers by attracting and supporting greater populations of avian seed dispersal agents, the number of juniper seedlings was more than 2-fold greater in stands with high mistletoe density compared with stands that had little or no mistletoe. Results suggest that the occurrence of a three-way interaction, in the presence of environmental variation (in this case, annual variation in juniper berry crops), may change the ecological roles of associated species. A conceptual model is presented to illustrate how the role of mistletoe may range from parasitic to mutualistic, while the role of avian seed dispersers may conversely range from mutualistic to parasitic, the latter by acting as vectors for the spread of mistletoe.     

Enhanced chrysene degradation by halotolerant Achromobacter xylosoxidans using Response Surface Methodology

Degradation of chrysene, a four ring High Molecular Weight (HMW) Polycyclic Aromatic Hydrocarbon (PAH) is of intense environmental interest, being carcinogenic, teratogenic and mutagenic. Multiple PAH degrading halotolerant Achromobacter xylosoxidans was isolated from crude oil polluted saline site. Response Surface Methodology (RSM) using Central Composite Design (CCD) of Bushnell-Haas medium components was successfully employed for optimization resulting 40.79% chrysene degradation on 4th day. The interactions between variables as chrysene and glucose concentrations, pH and inoculum size on degradation were examined by RSM. Under optimum conditions, A. xylosoxidans exhibited 85.96% chrysene degradation on 5th day. The optimum values predicted by RSM were confirmed through confirmatory experiments. It was also noted that pH and glucose as co-substrate play a dynamic role in enhancement of chrysene degradation. Hence, A. xylosoxidans can be further used for subsequent microcosm and in situ experiments for its potential to remediate PAH contaminated saline and non-saline soils.     

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.     

Studies revealing bioremediation potential of the strain Burkholderia sp. GB-01 for abamectin contaminated soils

Burkholderia sp. GB-01 strain was used to study different factors affecting its growth for inoculum production and then evaluated for abamectin degradation in soil for optimization under various conditions. The efficiency of abamectin degradation in soil by strain GB-01 was seen to be dependent on soil pH, temperature, initial abamectin concentration, and inoculum size along with inoculation frequency. Induction studies showed that abamectin depletion was faster when degrading cells were induced by pre-exposure to abamectin. Experiments performed with varying concentrations (2–160 mg Kg⁻¹) of abamectin-spiked soils showed that strain GB-01 could effectively degrade abamectin over the range of 2–40 mg Kg⁻¹. The doses used were higher than the recommended dose for an agricultural application of abamectin, taking in account the over-use or spill situations. A cell density of approximately 10⁸ viable cells g⁻¹ dry weight of soil was found to be suitable for bioremediation over a temperature range of 30–35°C and soil pH 7.5–8.5. This is the first report on bacterial degradation of abamectin in soil by a Burkholderia species, and our results indicated that this bacterium may be useful for efficient removal of abamectin from contaminated soils.