Production of a microbial lipase by Staphylococcus carnosus (pLipMut2) in a bubble column reactor and a centrifugal field bioreactor

Extracellular lipase production by the recombinant strain Staphylococcus carnosus (pLipMut2) has been studied. First substrate optimization was carried out in shaken cultures. As a result, the best substrate yield of 20 units/g (peptone + yeast extract) and maximum lipase activity in the culture supernatant of 1.7 units/cm³ could be obtained by a nutrient rich complex medium consisting of 75 kg/m³ yeast extract, 15 kg/m³ tryptone, 5 kg/m³ glucose and 0.5 kg/m³ K₂HPO₄. Higher initial substrate concentration caused inhibition of growth. Antifoam agent at higher levels than 1 cm³/ dm³ resulted in a negative influence on lipase yield. Comparative fermentation studies have been carried out in a bubble column reactor and in a centrifugal field bioreactor. Direct proportionality between growth, lipase production and oxygen consumption was observed. In the bubble column reactor usual superficial air velocities (4 cm/s) caused intensive foam generation, thus fermentation was only possible after installation of a broader column head to allow coalescence. In the centrifugal field bioreactor higher productivities were obtained without foam problems at superficial gas velocities which were one order of magnitude lower than in the bubble column. Fermentations have been performed batchwise and without holding pH constant. Neither pH control nor glucose feeding could improve the substrate yield further. Compared to former fermentation studies with the strain S. carnosus (pLipPS1) lipase yield (lipase activity/cell density) could be improved by 300% and substrate yield (lipase activity/substrate concentration) by 600%.     

Incorporating hydrodynamics into spatiotemporal metabolic models of bubble column gas fermentation

Gas fermentation has emerged as a technologically and economically attractive option for producing renewable fuels and chemicals from carbon monoxide (CO) rich waste streams. LanzaTech has developed a proprietary strain of the gas fermentating acetogen Clostridium autoethanogenum as a microbial platform for synthesizing ethanol, 2,3-butanediol, and other chemicals. Bubble column reactor technology is being developed for the large-scale production, motivating the investigation of multiphase reactor hydrodynamics. In this study, we combined hydrodynamics with a genome-scale reconstruction of C. autoethanogenum metabolism and multiphase convection–dispersion equations to compare the performance of bubble column reactors with and without liquid recycle. For both reactor configurations, hydrodynamics was predicted to diminish bubble column performance with respect to CO conversion, biomass production, and ethanol production when compared with bubble column models in which the gas phase was modeled as ideal plug flow plus axial dispersion. Liquid recycle was predicted to be advantageous by increasing CO conversion, biomass production, and ethanol and 2,3-butanediol production compared with the non-recycle reactor configuration. Parametric studies performed for the liquid recycle configuration with two-phase hydrodynamics showed that increased CO feed flow rates (more gas supply), smaller CO gas bubbles (more gas–liquid mass transfer), and shorter column heights (more gas per volume of liquid per time) favored ethanol production over acetate production. Our computational results demonstrate the power of combining cellular metabolic models and two-phase hydrodynamics for simulating and optimizing gas fermentation reactors.     

Chlortetracycline production with immobilized Streptomyces aureofaciens

Summary The semicontinuous production of chlortetracycline by immobilized cells of Streptomyces aureofaciens ATCC 10762 was compared with that of free cells. Immobilized cells transferred repeatedly to a new production medium, showed a fourfold increase in the half life time of antibiotic production.In an air bubble column a high chlortetracycline productivity was obtained with a high aeration rate.A semicontinuous production of chlortetracycline by immobilized S. aureofaciens could be improved by varying the fermentation conditions.For continuous chlortetracycline production by immobilized cells, no improvement was detected.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

High yield <Emphasis Type="Italic">Rhizopus chinenisis</Emphasis> prolipase production in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: Impact of methanol concentration

Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5 to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L, with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol fed-batch phase was very important for cell viability and protease activity.     

Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor

 Cultivation of Monascus purpureus (CCRC 31615) for the production of natural pigments was investigated. Traditionally, Monascus species were grown on rice by solid-state culture. For large-scale cultivation, solid-state cultures were associated with some problems such as contamination and scale-up. By using submerged cultures with rice particles, a stirred-tank fermentor was not suitable for submerged cultures as the impeller tended to break the particles into small pieces. A conventional bubble column was also unsuitable as its mixing capability was poor. In the present study, a modified bubble column with wire-mesh draft tubes was employed for the cultivation of M. purpureus. The proposed column had a shorter mixing time and a higher oxygen transfer rate relative to the conventional bubble column. The production of pigments using the proposed column was up to 80% higher than that achieved using the conventional bubble column. Received: 21 July 1999 / Received revision: 8 November 1999 / Accepted: 19 November 1999     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Production and regulation of different lipase activities from Rhizopus chinensis in submerged fermentation by lipids

The fungal Rhizopus chinensis could produce several types of lipase, which were mainly intracellular. During the whole-cell lipase production by this strain in submerged fermentation, it was observed that two catalytic characteristics (hydrolytic and synthetic activity) of lipases were different with addition of lipids. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. However, it was found that the induction of lipids for the synthetic activity lipase was significant, and that nearly no synthetic activity was detected while the medium contained no lipids. When only a little lipid (1 g/L) was added to medium, the synthetic activity increased sharply in the initial process of fermentation. Analysis of crude membrane-bound lipase by SDS-PAGE confirmed this induction. De novo biosynthesis of lipases, especially the lipase with synthetic activity occurred only when lipids existed. Cell growth and maltose repress the lipase production with synthetic activity, but have little influence on the lipase production with hydrolytic activity. Since the production process of mycelium-bound lipase with hydrolytic activity was different, it was reasonable to consider hydrolytic activity and synthetic activity for different application purposes. Whole-cell lipase obtained from fermentation process with high synthetic activity showed excellent catalytic ability in solvent free system on synthesis of ethylcaprylate and ethyloleate, the conversion could reach more than 90% in 5 h.     

Xanthomonas campestris as a host for the production of recombinantPseudomonas aeruginosa lipase

Recombinant plasmid pBP13, which expresses the alkaline lipase fromPseudomonas aeruginosa IGB83 under thetac promoter was transferred toXanthomonas campestris pvcampestris IBT148. Different fermentation conditions were tested for lipase productivity by strain IBT148 carrying plasmid pBP13, and a fermentation process was established in an instrumented bioreactor, where lipase production was increased more than 12-fold with respect to the initial culture conditions in shake flasks. Xanthan gum stabilized the activity of the alkaline lipase.     

Changes in morphology of <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> in submerged fermentation and their effect on production of mycelium-bound lipase

In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.     

Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

On-line monitoring of lipase production in fermentation processes

Summary The results obtained in on-line monitoring of lipase production byCandida rugosa in a batch fermentation process are presented. For this purpose an automatic lipase concentration analyzer has been developed and tested. Its good reproducibility and its capability to perform the analysis in less time than the classical titrimetric method make it very suitable for on-line determination lipase concentration in fermentation processes.     

Transgenic rice as bioreactor for production of the Candida antarctica lipase B

Candida antarctica lipase B (CALB) is a versatile biocatalyst used for a wide range of biotransformation. Methods for low cost production of this enzyme are highly desirable. Here, we report a mass production method of CALB using transgenic rice seeds as the bioreactor. The transgenic rice transformed with the CALB gene under the control of the promoter of the rice seed storage protein GT1 was found to have accumulated a large quantity of CALB in seeds. The transgenic line with the highest lipolytic activity reached to 85 units per gram of dry seeds. One unit is defined as the amount of lipase necessary to liberate 1 μmol p‐nitrophenol from p‐nitrophenyl butyrate in 1 min. The rice recombinant lipase (rOsCALB) from this line represents 40% of the total soluble proteins in the crude seed extracts. The enzyme purified from the rice seeds had an optimal temperature of 40 °C, and optimal pH of 8.5, similar to that of the fermentation products. Test of its conversion ability as a biocatalyst for biodiesel production suggested that rOsCALB is functionally identical to the fermentation products in its industrial application.     

Hydrodynamics in bubble column bioreactors with fermentation broths having a yield stress

Summary The hydrodynamics in a bubble column bioreactor with fermentation broths having a yield stress are studied. Specifically, the liquid velocity at the reactor axis, the axial dispersion coefficient, and the gas hold-up are examined. The liquid velocity at the reactor axis and the gas hold-up are measured in a 40-1 bench-scale bubble column fermentor using carboxypolymethylene (Carbopol) aqueous solutions as simulated broths. Theoretical correlations for the liquid velocity at the reactor axis, the axial dispersion coefficient, and the gas hold-up are derived on the basis of an energy balance and the mixing length theory. The correlations are compared with the present data and a reasonable agreement is found. The theoretical predictions are also in satisfactory agreement with the re-examined data for actual fermentation broths which are Chaetomium cellulolyticum and Neurospora sitophila cultured in a 1000-1 pilot-plant scale airlift fermentor.     

Production of ethanol and biomass from thin stillage by Neurospora intermedia: A pilot study for process diversification

Dry mill ethanol processes produce ethanol and animal feed from whole grains, where the wastewater after the distillation and separation of solid materials is called “thin stillage.” In this work, similar production of ethanol (3.5 g/L) and biomass (5 g/L) from thin stillage was obtained during batch cultivation of the edible fungus Neurospora intermedia in a 2‐m high airlift reactor and bubble column. The fungal biomass, containing 50% w/w protein and 12% w/w lipids, was rich in essential amino acids and omega‐3 and ‐6 fatty acids. In a continuous mode of fermentation, dilution rates of up to 0.2 h^?1 could be applied without cell washout in the bubble column at 0.5 vvm. At 0.1 h^?1, around 5 g/L of ethanol and 4 g/L of biomass containing ca. 50% w/w protein were produced. The fungus was able to assimilate saccharides in the liquid fraction as well as sugar backbones such as xylan and arabinan in the solid fraction. The inclusion of the current process could potentially lead to the production of 11 000 m³ of ethanol (5.5% improvement vs. normal industrial process) and around 6300 tons of high‐quality biomass for animal feed at a typical facility producing 200 000 m³ ethanol per year.     

Xanthan batch fermentations: compared performances of a bubble column and a stirred tank fermentor

Fermentations of Xanthomonas campestris, NRRL B-1459, were carried out in a bubble column fermentor (BCF) and in a stirred tank fermentor (STF) to allow comparison of representative variables measured during the microbial growth and the gum production. The microbial growth phase was described by a logistic rate equation where maximum cell concentration was provided by nitrogenous compounds balance. The average value of the maximum specific growth rate was higher in the bubble column (μ _M =0.5 h^?1) than in the stirred reactor (μ _M =0.4 h^?1). The upper values of xanthan yield (Y _g-x =0.65 kg xanthan/kg glucose; Y _{O 2?x} xanthan/kg oxygen) and specific production rate (q _x =0.26 kg xanthan/kg biomass · h) were measured when the oxygen transfer coefficient was kept up above 80 h^?1 in the STF fermentor. In the bubble column the fermentation achieved in the same culture medium lasts two times longer than in the stirred aerated tank; this was attributed to the low value of the oxygen transfer coefficient (K _L a =20 h^?1) at the beginning of the gum synthesis phase. The results obtained in the stirred tank were the basis to estimate the optimal biomass concentration which enables to achieve a culture in non-limiting oxygen transfer conditions. Nevertheless, the transfer characteristics were more homogeneous in the bubble column than in the stirred tank where dead stagnant zones were observed. This is of primary importance when establishing fermentation kinetics models.     

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress

The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS) during carotene production from waste cooking oil (WCO) and corn steep liquor (CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated. The specific activities of the intracellular enzymes superoxide dismutase (SOD) and catalase (CAT) as well as the micromorphology of the fungus were measured in order to study the response of the fungus to oxidative stress. The changes of the morphology of microorganism leaded to pellets formation and documented using a computerized image analysis system. As a consequence of the mild oxidative stress induced by hydroperoxides of WCO and ROS a significant increase in carotene production was obtained. The highest carotene concentration (980.0 mg/l or 51.5 mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO (50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the carotenes produced consisted of β‐carotene (71%), γ‐carotene (26%), and lycopene (3%). The strong oxidative stress in the fungus caused a significant increase of γ‐carotene concentration. Bubble column reactor is a useful fermentation system for carotene production in industrial scale.     

Lipase production and Penicillium simplicissimum morphology in solid-state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.     

Response surface method to optimize the production and characterization of lipase from Penicillium verrucosum in solid-state fermentation

Current studies about lipase production by solid-state fermentation involve the use of agro-industrial residues towards developing cost-effective systems directed to large-scale commercialization of enzyme-catalyzed processes. In this work, lipase production and partial characterization of the crude enzymatic extracts obtained by Penicillium verrucosum using soybean bran as substrate was investigated. Different inductors were evaluated and the results showed that there is no influence of this variable on the lipase production, while temperature and initial moisture were the main factors that affected enzyme production. The optimized cultivation temperature (27.5 °C) and initial moisture of substrate (55%) were determined using the response surface methodology. Kinetics of lipase production was followed at the optimized growth conditions. Optimum lipase yield was 40 U/g of dry bran. The crude enzymatic extract showed optimal activity in the range from 30 to 45 °C and in pH 7.0.