Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).     

Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides

Purpose of work

To establish pilot scale bioreactor cultures of somatic embryos of Siberian ginseng for the production of biomass and eleutherosides. Somatic embryos of Eleutherococcus senticosus were cultured in airlift bioreactors using Murashige and Skoog medium with 30 g sucrose l^?1 for the production of biomass and eleutherosides. Various parameters including the type of bioreactor, aeration volume, and inoculum density were optimized for 3 l capacity bioreactors. Balloon-type airlift bioreactors, utilizing a variable aeration volume of 0.1–0.3 vvm and an inoculum of 5 g l^?1, were suitable for biomass and eleutheroside production. In 500 l balloon-type airlift bioreactors, 11.3 g dry biomass l^?1, 220 µg eleutheroside B l^?1, 413 µg eleutheroside E l^?1, and 262 µg eleutheroside E1 l^?1 were produced.     

Optimization of culturing conditions for production of somatic embryos and lignins of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill

Schisandra chinensis (Turcz.) Baill. is a valuable medicinal plant species increasingly used in phytotherapy worldwide. This study systematically detected the lignin content and production during somatic embryogenesis of S. chinensis. The effect of various culture parameters on biomass accumulation and lignin production were also examined to optimize the accumulation of lignins in SEs in bioreactors, including the culture method, inoculum density, aeration volume and photoperiod. An inoculum density of 20 g L^??1 embryogenic calli enhanced production of lignin, while 30 g L^??1 embryogenic calli increased the biomass of somatic embryos. During somatic embryo induction, an aeration volume of 0.2 vvm and photoperiod of 16 h day^??1 were found to be optimal for biomass accumulation and lignin production. An approximately threefold increase in the biomass production rate and a fourfold increase in the total lignin production rate in SEs were achieved in bioreactors than on solid medium. The present study indicated, therefore, that the culturing of S. chinensis somatic embryos in bioreactors is an effective method for the industrialized production of lignin in vitro.     

Lipid production and composition of fatty acids in Chlorella vulgaris cultured using different methods: photoautotrophic,heterotrophic, and pure and mixed conditions

This study investigated the biomass, lipid production, fatty acid content, and other nutrients present in microorganisms by using four culture methods: (1) photoautotrophic pure Chlorella vulgaris cultures (PP); (2) heterotrophic pure C. vulgaris cultures (PH); (3) mixed cultures of Rhodotorula glutinis and C. vulgaris under photoautotrophic conditions (MP); and (4) heterotrophic mixed cultures (MH). The microorganisms in MP culture showed the optimum growth condition and lipid production. Among the cultures, MP yielded the highest number of cells and biomass (5.9?×?10⁵ cells/mL and 0.523 g/L, respectively). Furthermore, lipid production in MP culture was 114.22 mg/L, which is 136 % higher than that in MH culture (48.22 mg/L). Considering the higher contents of palmitic acid (C16:0) at 24.65 %, oleic acid (C18:1) at 56.34 %, and protein at 42.39 g/100 g in the MP culture than in other cultures, we proposed that MP could be used effectively to support the growth of microorganisms. This method could also be used as a potential approach for biodiesel production.     

Screening of phytase producers and optimization of culture conditions for submerged fermentation

Phytase (myo-inositol-hexakisphosphate phosphohydrolase) is an enzyme, which breaks down phytate to inositol and orthophosphoric acid. Phytase has been used as feed additive, and in some medical applications for years. To date, phytase production has been usually performed as a solid-state fermentation with small production volumes. Therefore, the aim of this study was to increase the phytase activity in submerged fermentations by screening several microorganism strains based on the literature to select the most productive phytase producer and optimizing growth parameters such as temperature, pH, and aeration level using response surface methodology (RSM). As a result, among the four different microorganisms evaluated, Aspergillus ficuum (NRRL 3135) was selected as the most productive strain. Optimum temperature, pH, and aeration values were determined as 33 °C, 4.5, and 0.9 vvm, respectively, for A. ficuum in 2-l batch submerged phytase productions. Under these conditions, phytase activity was measured as 2.27 U/ml. Therefore, this is a unique study showing the production of phytase with A. ficuum successfully in submerged fermentation as opposed to the traditional solid-state fermentation.     

Influence of inoculum density and aeration volume on biomass and bioactive compound production in bulb-type bubble bioreactor cultures of Eleutherococcus koreanum Nakai

This study deals with the effects of initial inoculum density and aeration volume on biomass and bioactive compound production in adventitious roots of Eleutherococcus koreanum Nakai in bulb-type bubble bioreactors (3-L capacity). While the fresh and dry weights of the roots increased with increasing inoculum density, the highest percentage dry weight and accumulation of total target compounds (eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids) were noted at an inoculum density of 5.0 g L⁻¹. Poor aeration volume (0.05 vvm) stunted root growth, and high aeration volume (0.4 vvm) caused physiological disorders. Moreover, an inoculum density of 5.0 g L⁻¹ and an aeration volume of 0.1 vvm resulted in the highest concentration of total target compounds and least root death. Such optimization of culture conditions will be beneficial for the large-scale production of E. koreanum biomass and bioactive compounds.     

Enhanced Production of carboxymethylcellulase by a marine bacterium,Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.     

Cyclodextrin glycosyltransferase production by free cells of <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R in batch fermentation and by immobilized cells in a semi-continuous process

Cyclodextrin glycosyltransferase (CGTase) catalyzes starch conversion into cyclic or linear oligosaccharides, important industrial products for the complexation of non-polar substances. In this work, conditions to increase CGTase production from Bacillus circulans strain DF 9R were optimized by two systems. On one hand, free cells were grown in batch fermentation experiments to optimize aeration and pH. The highest activity (1.47 ± 0.21 U ml^?1) was achieved after 48 h of growth, aeration of 1.5 vvm and pH regulated to 7.6. On the other hand, bacterial cells were immobilized on loofa and synthetic sponge, and used for CGTase production in a semi-continuous process. An initial biomass of 30 mg of lyophilized cells and an immobilization time of 24 h with loofa or synthetic sponge were enough to achieve increased production of CGTase: 0.91 ± 0.10 and 0.95 ± 0.11 U ml^?1, respectively. Sponges with immobilized bacteria were reused in 12 successive cycles. Besides, in our conditions, CGTase was not adsorbed onto the supports used for immobilization, which ensured the total recovery of the enzyme from the culture medium. The two CGTase production processes studied showed similar productivity and could be potentially scaled up.     

Itaconic acid production from xylose in repeated-batch and continuous bioreactors

Summary Itaconic acid production from xylose by immobilized Aspergillus terreus TKK 200-5-2 mycelia was optimized both in repeated shake-flask fermentations and in continuous column bioreactors using statistical experimental design and empirical modelling. Using continuous 9-1 scale air-lift bioreactors, a pH of 2.5, aeration rate of 0.6 v/v per minute and residence time of 160 h gave the highest itaconic acid concentration. In air-lift bioreactors a cubic carrier size of 0.5 cm gave a 3.3-fold higher product concentration than 1-cm cubes. Packed-bed column reactors had a higher production rate than air-lift reactors. Offprint requests to: H. Kautola     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

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.     

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high β-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Microbial production of 2,3-butanediol by a newly-isolated strain of Serratia marcescens

A newly-isolated strain of Serratia marcescens, G12, was characterized for 2,3-butanediol (2,3-BD) production. In shake-flask and batch fermentations, 2,3-BD reached 48.5 and 51 g l^?1, respectively. Low amounts of (~8 g l^?1) of acetoin were also formed. In fed-batch fermentations, strain G12 produced 72.8 g 2,3-BD l^?1 with glucose initially at 130 g l^?1. When aeration rate was increased to 2.5 vvm for the fermentation process, 2,3-BD reached 87.8 g l^?1 and the highest productivity was 1.6 g l^?1 h^?1. Acetoin was at 6.2 g l^?1. G12 therefore may be a suitable candidate strain for large-scale production of 2,3-BD.     

Optimized aeration by carbon dioxide gas for microalgal production and mass transfer characterization in a vertical flat-plate photobioreactor

To optimize the aeration conditions for microalgal biomass production in a vertical flat-plate photobioreactor (VFPP), the effect of the aeration rate on biomass productivity was investigated under given conditions. Air enriched with 5% or 10% (v/v) CO(2) was supplied for the investigation at rates of 0.025-1 vvm. The CO(2) utilization efficiency, change of pH in the medium, and the optimum aeration rate were determined by evaluating biomass productivity. To investigate the VFPP mass transfer characteristics, the overall volumetric mass transfer coefficient, k(L)a, was evaluated for several different flat-plate sizes. Increasing the height of the VFPP could improve both the mass transfer of CO(2) and the illumination conditions, so this appeared to be a good method for scaling up. Based on a comparison of the k(L)a value at the optimum aeration rate with previously reported results, it was confirmed that the range of CO(2) concentration used in the experiments was cost-effective for mass culture.     

Production of protease using wastewater from the manufacture of shochu

The production of protease using wastewater from a shochu distillery was investigated in order to devise a process for the treatment of shochu distillery wastewater. Aspergillus usami mut. shirousami IFO 6082 was selected from among eight strains for production of protease. Production of 240 U/ml of proteolytic activity was achieved after 72 h in a jar-fermentor culture under the following conditions: initial pH, 5; 30°C; aeration, 1 vvm; and agitation, 600 rpm. The protease was purified by column chromatography on Sephadex G-150 and isoelectrofocusing. The molecular weight of the purified enzyme determined by SDS-PAGE was 56 kDa, and the isoelectric point was pH 4.0. The optimum pH for the reaction was about 4.0, so the enzyme is therefore an acid protease. The optimum temperature for proteolysis ranged between 55 and 60°C, while the enzyme was unstable at temperatures above 60°C.     

Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia (L.) in a liquid-phase airlift balloon-type bioreactor

To improve root growth and production of bioactive compounds such as anthraquinones (AQ), phenolics, and flavonoids by adventitious root cultures of Morinda citrifolia, the effects of aeration rate, inoculum density, and Murashige and Skoog (MS) medium salt strengths were investigated using a balloon-type bubble bioreactor. The possible mechanisms underlying changes in activities of enzymic (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase) and nonenzymic (vitamin E) antioxidants, phenylalanine ammonia lyase, and stress levels (accumulation of hydrogen peroxide and proline, peroxidation of lipids) were also studied. Low aeration rate (0.05 vvm [air volume/culture volume/min]) accelerated accumulation of root fresh weight and dry weight (DW). High aeration rates (0.1 to 0.3 vvm) stimulated accumulation of AQ, phenolics, and flavonoids and reduced root growth. Low inoculum densities (5 and 10 g l^–1) increased accumulation of those metabolites but inhibited root growth. Culture of adventitious roots with high concentrations of MS salts (1× and 1.5× MS) resulted in induction of oxidative stress that strongly inhibited root growth. Overall, an aeration rate of 0.05 vvm, 15 g l^–1 inoculum density, and half-strength (0.5×) MS medium were optimal for enhancing accumulation of root dry biomass (4.38 g l^–1), AQ (103.08 mg g^–1 DW), phenolics (54.81 mg g^–1 DW), and flavonoids (49.27 mg g^–1 DW).     

松茸液态发酵菌丝生长量因子的研究

研究了液态发酵条件下通气量、转速、pH、可溶性淀粉浓度对松茸菌丝生长量的影响,探讨了可溶性淀粉稳定pH的作用机理。试验结果表明：通气量≤0.4vvm时松茸菌丝产量随通气量的增大而增长,当通气量＞0.5vvm时,菌丝产量增加不明显;转速增大菌丝产量增加,转速＞80r/min,菌丝产量下降;松茸菌丝发酵的最适pH为5.5,初始pH不同,发酵罐中培养基最终的pH趋于6.0;可溶性淀粉是影响松茸菌丝量的重要因子,可溶性淀粉浓度≤4g/100mL时菌丝量随可溶性淀粉浓度的增加而增大,当其浓度为4g/100mL时,菌     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.