Studies of some physical parameters for large scale protease production by SSF

For designing the large scale protease production system, through solid state fermentation, the important parameters are accumulation density, bed height, aeration etc. The optimum fermentation condition recorded were 0.3125 g/cc accumulation density and 1.0 cm bed height (without aeration). Aeration at 3 LPM and 2.0 cm bed height were found optimum for protease production by Rhizopus oryzae.     

Optimization of some additives to improve protease production under SSF

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.     

A short review on SSF - an interesting process option for ethanol production from lignocellulosic feedstocks

Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37 degrees C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L-1 with a yield based on total hexoses and pentoses higher than 70%.     

Accelerated process development for protease production in continuous multi-stage cultures

A fermentation process was developed and optimized for the production of a specific protease from Bacillus licheniformis PWD-1. Media formulations were constructed and crucial environmental parameters were optimized to enhance growth and product formation. Process dynamics of substrate consumption, biomass-, product-, as well as by-product formation were determined under controlled conditions in a bioreactor. Using kinetic data from batch- and continuous-culture experiments, a fed-batch process was developed producing proteolytic activities 10 times those found during regular batch culture. In one stage continuous stirred tank culture protease formation was completely decoupled from sporulation. Shift experiments in one-stage continuous cultures led to the development of a two-stage continuous stirred tank fermentation process using optimized conditions for growth in the first stage and protease formation in the second stage. Accordingly, the basis for a continuous production of the enzyme on a pilot scale was accomplished.     

Integrating sugarcane molasses into sequential cellulosic biofuel production based on SSF process of high solid loading

Background

Sugarcane bagasse (SCB) is one of the most promising lignocellulosic biomasses for use in the production of biofuels. However, bioethanol production from pure SCB fermentation is still limited by its high process cost and low fermentation efficiency. Sugarcane molasses, as a carbohydrate-rich biomass, can provide fermentable sugars for ethanol production. Herein, to reduce high processing costs, molasses was integrated into lignocellulosic ethanol production in batch modes to improve the fermentation system and to boost the final ethanol concentration and yield.

Results

The co-fermentation of pretreated SCB and molasses at ratios of 3:1 (mixture A) and 1:1 (mixture B) were conducted at solid loadings of 12% to 32%, and the fermentation of pretreated SCB alone at the same solid loading was also compared. At a solid loading of 32%, the ethanol concentrations of 64.10 g/L, 74.69 g/L, and 75.64 g/L were obtained from pure SCB, mixture A, and mixture B, respectively. To further boost the ethanol concentration, the fermentation of mixture B (1:1), with higher solid loading from 36 to 48%, was also implemented. The highest ethanol concentration of 94.20 g/L was generated at a high solid loading of 44%, with an ethanol yield of 72.37%. In addition, after evaporation, the wastewater could be converted to biogas by anaerobic digestion. The final methane production of 312.14 mL/g volatile solids (VS) was obtained, and the final chemical oxygen demand removal and VS degradation efficiency was 85.9% and 95.9%, respectively.

Conclusions

Molasses could provide a good environment for the growth of yeast and inoculum. Integrating sugarcane molasses into sequential cellulosic biofuel production could improve the utilization of biomass resources.

     

Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 10⁵ U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 10⁵ U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 10⁵ U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].     

Some observations on protease production in continuous suspension cultures of Bacillus firmus

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. (c) 1993 John Wiley & Sons, Inc.     

Inoculation effect of thermophilic microorganisms on protease production through solid-state fermentation under non-sterile conditions at lab and bench scale (SSF)

The production of protease enzyme was evaluated through the solid state fermentation (SSF) of soy fibre, a waste product that acted as a sole substrate for the fermentation, at a laboratory and bench scale using a 500-mL (batch size 115 g) and 10-L (batch size 2300 g) bioreactors. The objective was to assess the effect of the inoculation of the thermophilic bacteria Thermus sp. on the production of the enzyme when working at laboratory and bench scale under non-sterile conditions, since scaling-up and the need of sterilization are the main challenges of SSF, preventing its industrial development. Results revealed that the inoculation led to a substantial increase in the protease obtained on both scales when compared to non-inoculated fermentation. The maximum protease activities increased as a result of the inoculation from 500 to 800 and from 350 to 670 U/g dry matter of soy fibre in the lab and bench scale bioreactors, respectively. Finally, a very good correlation was found between the protease activities obtained and the fermentation most relevant parameters: oxygen uptake rate (R ² = 0.81) and temperature (R ² = 0.82). In this work, we have demonstrated that inoculation is effective even under non-sterile conditions at the kg scale and that this strain is able to compete with autochthonous microbiota and increase the protease production to levels higher than those previously reported in literature.     

Alkaline protease production by an isolated Bacillus circulans under solid-state fermentation using agroindustrial waste: process parameters optimization

Alkaline protease production using isolated Bacillus circulans under solid-state fermentation environment was optimized by using Taguchi orthogonal array (OA) experimental design (DOE) methodology to understand the interaction of a large number of variables spanned by factors and their settings with a small number of experiments in order to economize the process optimization. The software-designed experiments with an OA worksheet of L-27 was selected to optimize fermentation (temperature, particle size, moisture content and pH), nutrition (yeast extract and maltose), and biomaterial-related (inoculum size and incubation time) factors for the best production yields. Analysis of experimental data using Qualitek-4 methodology showed significant variation in enzyme production levels (32,000-73,000 units per gram material) and dependence on the selected factors and their assigned levels. Validation of experimental results on alkaline protease production by this bacterial strain based on DOE methodology revealed 51% enhanced protease production compared to average performance of the fermentation, indicating the importance of this methodology in the evaluation of main and interaction effects of the selected factors individually and in combination for bioprocess optimization.     

SSF production of lactic acid from cellulosic biosludges

The use of cellulosic biosludges generated in a Kraft pulp mill was investigated as substrate for lactic acid production by simultaneous saccharification and fermentation (SSF). The effect of the operation mode (batch or fedbatch), the initial liquid to solid ratio (12 or 30 g/g) and the nutrient supplementation (MRS components or none) on several parameters including lactic acid concentration, volumetric productivity and product yields, were evaluated. When the operation was carried out in fedbatch mode with nutrient supplementation and using a LSR(0)=12 g/g, a broth containing 42 g/L was obtained after 48 h with a volumetric productivity of 0.87 g/L h and a product yield of 37.8 g lactic acid/100 g biosludges. In a similar experiment carried out without nutrient supplementation, a lactic acid concentration of 39.4 g/L was obtained after 48 h with a volumetric productivity of 0.82 g/L h and a product yield of 35.5 g L-lactic acid/100 g biosludges.     

Development and optimization of an adenovirus production process

Adenoviral vectors have a number of advantages such as their ability to infect post-mitotic tissues. They are produced at high titers and are currently used in 28% of clinical protocols targeting mainly cancer diseases through different strategies. The major disadvantages of the first generation of recombinant adenoviruses are addressed by developing new recombinant adenovirus vectors with improved capacity and safety and reduced inflammatory response. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. HEK-293 complementing cell line physiology, metabolism and viral infection kinetics were studied at small scale to identify optimal culture conditions. Batch, fed-batch and perfusion culture modes were evaluated. Development of new monitoring tools (in situ GFP probe) and quantification techniques (HPLC determination of total viral particles) contributed to acceleration of process development. On-line monitoring of physiological parameters such as respiration and biovolume of the culture allowed real-time supervision and control of critical phases of the process. Use of column chromatographic steps instead of CsCl gradient purification greatly eased process scale-up. The implementation of the findings at large scale led to the development of an optimized and robust integrated process for adenovirus production using HEK-293 cells cultured in suspension and serum-free medium. The two-step column-chromatography purification was optimized targeting compliance with clinical material specifications. The complete process is routinely operated at a 20-L scale and has been scaled-up to 100 L. Scale-up of adenoviral vector production in suspension and serum-free medium, and purification according to regulatory requirements, are achievable. To overcome metabolic limitations at high cell densities, use of perfusion mode with low-shear cell retention devices is now a common trend in adenovirus manufacturing. Further process improvements will rely on better understanding of the mechanisms of virus replication and maturation in complementing host cells.     

Media optimization studies for glucose isomerase production by arthrobacter species

Media optimization studies are carried out with the objective of maximising glucose isomerase production by Arthrobacter sp. The recommended media consists of 1.0% (w/v) xylose, 1.0% peptone, 0.5% yeast extract, 0.025% MgSO₄·7H₂O, 0.6% (NH₄)₂HPO₄ and 0.2% KH₂PO₄. Activity of the enzyme produced in this media is 11.2 units/ml. Growth cycle for batch cultivation is studied and the lag period is 2 hours, followed by exponential phase extending upto 32 hours.     

A process optimization study on ultrasonic extraction of paclitaxel from Taxus cuspidata

This study aimed to improve the extraction rate of paclitaxel from Taxus cuspidata in order to determine the most effective combination of ultrasonic extraction and thin-layer chromatography–ultraviolet (TLC-UV) rapid separation method. The study was performed using the Box–Behnken test design to conduct single-factor experiments using ultrasonic extraction of paclitaxel from Taxus cuspidata. The study showed ethanol to be the best extraction solvent. When mixed with dichloromethane (1:1), the ratio of material to liquid was 1:50 when using an ultrasonic time of 1 hr at a power of 200 W. The correction coefficient K for the separation and detection of paclitaxel using the TLC-UV spectrophotometric method was 0.009152. Multifactor experiments determined the effect of the rate of liquid to material (X1), ultrasonic time (X2), and ultrasonic power (X3) on extraction using extraction volume as the dependent variable. Response surface analysis allowed a regression equation to be obtained, with the optimal conditions for extraction when the rate of liquid to material was 53.23 mL/g as an ultrasonic time of 1.11 hr and an ultrasonic power of 207.88 W. Using these parameters, the average amount of extracted paclitaxel was about 130.576 µg/g, which was significantly better than for other extraction methods.     

Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation

Production of alkaline protease employing the laboratory isolate, Bacillus sp. under solid state fermentation (SSF) was optimized. The effect of wheat bran and lentil husk was examined. Wheat bran showed highest enzyme production. The appropriate incubation time, inoculum size, moisture level and buffer solution level were determined. Maximum yields of 429.041 and 168.640 U g⁻¹ were achieved by employing wheat bran and lentil husk as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10 with 30 and 40% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20 and 25% and 0.5:1 for wheat bran and lentil husk, respectively.     

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728?U?ml^?1), which was followed by gram husk (714?U?ml^?1), mustard cake (680?U?ml^?1), and soybean meal (653?U?ml^?1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020?U?ml^?1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.     

Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics

Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G‐CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP‐HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355–362, 2016     

Some studies on the protease from a novel source: The plantCucurbita ficifolia

Summary An alkaline protease from the plantCucurbita ficifolia was studied using corn gluten as a substrate. At 25 units/ml, the enzyme solubilized 61% of the initial insoluble protein in 18.5 hours at pH 7.0, 55°C and 100 g/l of substrate.     

L-苏氨酸工业生产发酵条件优化研究

发酵法生产L-苏氨酸是目前广泛采用的方法,因此研究工业生产发酵条件优化具有重要意义。试验以高产L-苏氨酸菌作为出发菌株,结合本公司的实际工业生产条件对发酵各条件进行了一系列优化研究,结果表明：添加0.2%的工业级生长促进剂,以复合糖代替葡萄糖为初糖,并控制初糖浓度在60g/L,除生长高峰期外,发酵过程中溶解氧（DO）控制在10%~20%之间;最终发酵放罐湿菌体在45g/L左右,L-苏氨酸含量可达110g/L左右。     

Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization

A triphasic process was developed for the production of beta dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE(al)) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter(-1) citrate, pH 6.5, and 37 degrees C are ideal parameters for CphE(al) production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter(-1) CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter(-1) induced CphE(al) production with only about 30% efficiency in comparison to that with CGP. CphE(al) was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE(al) turned out to be a serine protease with maximum activity at 50 degrees C and at pH 7 to 8.5. Phase III comprises degradation of CGP to beta-aspartate-arginine and beta-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE(al) preparation. Optimum degradation parameters were 100 g liter(-1) CGP, 10 g liter(-1) crude CphE(al) powder, and 4 h of incubation at 50 degrees C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE(al) powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.