Production of glucose–fructose oxidoreductase and ethanol by Zymomonas mobilis ATCC 29191 in medium containing corn steep liquor as a source of vitamins

Different concentrations of corn steep liquor (CSL) were tested in the cultivation of Zymomonas mobilis. Cell growth, ethanol production, and the formation of glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL), the enzymes responsible for the bio-production of gluconic acid and sorbitol, were examined. The cell yields using 25 g CSL l(-1) and 40 g CSL l(-1) (Y(X,S) approximately 0.031 g g(-1)) were close to that obtained with 5 g yeast extract (YE) l(-1). With 5 g CSL l(-1) and 15 g CSL l(-1), the nutritional limitation led to smaller Y(X/S). Using 100 g CSL l(-1) produced an inhibitory effect on cell growth. Similar ethanol yields (92-95%) were calculated for each concentration of CSL and also for YE medium. The highest specific GFOR/GL activities (13.2-13.5 U g(-1) dry cell) were reached with 25 g CSL l(-1) and 40 g CSL l(-1), values comparable to that achieved with 5 g YE l(-1). The results confirm that CSL is an effective and cheap supplement for Z. mobilis medium, increasing the economic potential of a large-scale bio-production of sorbitol and gluconic acid by untreated Z. mobilis cells. The economic feasibility of the process is discussed.     

Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract

The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more ethanol (1.7 g L⁻¹) was produced with 20 g L⁻¹ CSL media in 250-mL bottle fermentations compared to media with 1 g L⁻¹ YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a 7.5-L fermentor with 10 and 20 g L⁻¹ CSL media were 8.6 and 9.6 g L⁻¹, respectively, which represent 57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L⁻¹ of ethanol was obtained in the medium with 1 g L⁻¹ YE after 360 h, which represents 53% of the theoretical ethanol yield from CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermentations. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11.     

A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Acetate production byClostridium thermoaceticum in corn steep liquor media

A low-cost nutrient medium based on corn steep liquor (CSL) was developed for the production of acetates byClostridium thermoaceticum. Pre-treatment of CSL with dolime and vitamin supplementation increased the rate of acetate production. Adding excess nutrients in a fed-batch mode minimized by-product formation and increased final acetate concentration from 19 g L^–1 to 40 g L^–1 acetic acid. High yields of acetic acid (0.95 g g^–1 glucose in fed-batch mode) was probably due to the conversion of the lactic acid in CSL into acetic acid by the organism.     

玉米浆对Vc二步发酵产2-酮基-L-古龙酸影响的研究

通过在培养基中添加不同量的玉米浆,研究其对氧化葡萄糖酸杆菌（俗称小菌）生产Vc前体2-酮基-L-古龙酸的影响,并研究玉米浆成分中的12种主要氨基酸对小菌产酸的影响。结果表明：每100 mL发酵培养基中添加2.5 g左右过滤除菌玉米浆时,2-酮基-L-古龙酸产量高达26.84 mg/mL,小菌活菌数为不添加玉米浆时小菌单菌发酵下的9.74倍。过量玉米浆抑制小菌产酸。12种氨基酸单独与氧化葡萄糖酸杆菌发酵培养及全部混合后与氧化葡萄糖酸杆菌发酵培养对产酸及菌体生长无影响。     

Development of Bacterial Contamination during Production of Yeast Extracts

Baker’s yeast suspensions having bacterial populations of 10⁶ and 10⁸ CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.     

Production of d-lactic acid by Lactobacillus delbrueckii ssp. delbrueckii from orange peel waste: techno-economical assessment of nitrogen sources

In this study, the effect of several organic nitrogen sources (namely peptone, meat extract—ME, yeast extract—YE, and corn steep liquor—CSL) on d-lactic acid production by Lactobacillus delbrueckii ssp. delbrueckii has been studied. While lactic acid bacteria (LAB) are well-known for their complex nutritional requirements, organic nitrogen source-related cost can be as high as 38% of total operational costs (OPEX), being its nature and concentration critical factors in the growth and productivity of the selected strain. Corn steep liquor (CSL) has been chosen for its adequacy, on the grounds of the d-lactic acid yield, productivity, and its cost per kilogram of product. Finally, orange peel waste hydrolysate supplemented with 37 g/l CSL has been employed for d-lactic acid production, reaching a final yield of 88% and a productivity of 2.35 g/l h. CSL cost has been estimated at 90.78$/ton of d-lactate.

     

Breeding of high ergosterol-producing yeast strains

High ergosterol-producing yeast strains YEH-28 and YEH-56 were constructed by hybridization of two haploids with opposite mating types from different species. The fermentation conditions of hybrid strain YEH-56 were studied. The highest level of ergosterol was obtained in 30 h at 28°C and 200 rpm, when 60 ml of culture in 250-ml shake flasks was grown in fermentation medium that consisted of (per liter): 100 g glucose; 10 g peptone and 10 g yeast extract. The initial pH was adjusted to 6.0. The optimal inoculum volume was 10% (v/v). Under optimal conditions, the yield of ergosterol of YEH-28 strain is 1.96 and 1.56 times that of the parental strains YE39 and YE244, whereas that of YEH-56 is 1.98 and 1.57 times that of the parental strains YE39 and YE244, respectively. Analysis of genetic stability showed that hybrid strains YEH-28 and YEH-56 are stable genetically. Journal of Industrial Microbiology & Biotechnology (2000) 25, 39–44. Received 13 November 1999/ Accepted in revised form 09 March 2000     

Production of chitinolytic enzymes by a strain (BM17) of Paenibacillus pabuli isolated from crab shells samples collected in the east sector of central Tyrrhenian Sea

Nineteen bacterial isolates were grown in shaken cultures in media containing chitin as carbon source and different additional nitrogen sources such as yeast nitrogen base (YNB), yeast extract (YE), corn steep liquor (CSL) and ammonium sulfate. Strain BM17 showed the highest activity (200 U/l) in medium containing Chitin (1%) and YNB (0.5%). Molecular analysis of the 16S rRNA gene showed that strain BM17 belongs to the species Paenibacillus pabuli (99.72% homology). The enzyme activity started after 12-24 h; exponential enzyme production was recorded from the 24th h and lasted till the 96th h of incubation when activity peaked to decrease thereafter. Medium optimisation was carried out by Response Surface Methodology (RSM) considering the effects of chitin, corn steep liquor and yeast extract. BM17 chitinolytic activity was induced by chitin but the increase of its concentration did not have significant effects on the enzyme activity. By contrast, the nitrogen source, particularly YE, strongly affected the enzyme production.     

Statistical optimization for production of carboxymethylcellulase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> DL-3 by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/DL-3 from rice bran using response surface method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.     

L-lactic acid production by Lactobacillus casei fermentation with corn steep liquor-supplemented acid-hydrolysate of soybean meal

Batch and fed-batch fermentation studies were performed to evaluate the potential of corn steep liquor (CSL)-supplemented acid-hydrolysate of soybean meal (AHSM) as an alternative to yeast extract (YE) for the production of L-lactic acid by Lactobacillus casei LA-04-1. The CSL-supplemented AHSM gave an outstanding result in supporting L-lactic acid production from glucose. In the exponential fed-batch fermentation, the concentration, yield and productivity of L-lactic acid were 162.5 g/L, 89.7% and 1.69 g/L per h, respectively, which were lower than those with 20 g/L YE (180 g/L, 90.3%, 2.14 g/L per h) after 96 h of fermentation. However, the raw material cost of the nitrogen resource was estimated as only 25% of that using the YE.     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Development of bacterial contamination during production of yeast extracts.

Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.     

Effect of corn steep liquor on xanthan production by Xanthomonas campestris

Summary The addition of corn steep liquor (CSL) to batch cultures of Xanthomonas campestris using sucrose as carbon source stimulated cell growth rate, viscosity and xanthan production as compared to non-supplemented cultures. The addition of CSL to a basal medium at a dose of 1 g/l, increased xanthan production and viscosity by 22% and 44% respectively. CSL also shortened the cultivation time and promoted a more efficient sucrose utilization for polymer synthesis. After 72 h of incubation the xanthan yield per sucrose consumed in the CSL-amended culture was 0.63 g/g, this is, 15% higher than without CSL addition. At higher doses of CSL cell growth rate was also increased but not polymer production.     

Optimization of a corn steep medium for production of ethanol from synthesis gas fermentation by <Emphasis Type="Italic">Clostridium ragsdalei</Emphasis>

Fermentation of biomass derived synthesis gas to ethanol is a sustainable approach that can provide more usable energy and environmental benefits than food-based biofuels. The effects of various medium components on ethanol production by Clostridium ragsdalei utilizing syngas components (CO:CO₂) were investigated, and corn steep liquor (CSL) was used as an inexpensive nutrient source for ethanol production by C. ragsdalei. Elimination of Mg²⁺, NH₄ ⁺ and PO₄ ³⁻ decreased ethanol production from 38 to 3.7, 23 and 5.93 mM, respectively. Eliminating Na⁺, Ca²⁺, and K⁺ or increasing Ca²⁺, Mg²⁺, K⁺, NH₄ ⁺ and PO₄ ³⁻ concentrations had no effect on ethanol production. However, increased Na⁺ concentration (171 mM) inhibited growth and ethanol production. Yeast extract (0.5 g l⁻¹) and trace metals were necessary for growth of C. ragsdalei. CSL alone did not support growth and ethanol production. Nutrients limiting in CSL were trace metals, NH₄ ⁺ and reducing agent (Cys: cysteine sulfide). Supplementation of trace metals, NH₄ ⁺ and CyS to CSL (20 g l⁻¹, wet weight basis) yielded better growth and similar ethanol production as compared to control medium. Using 10 g l⁻¹, the nutritional limitation led to reduced ethanol production. Higher concentrations of CSL (50 and 100 g l⁻¹) were inhibitory for cell growth and ethanol production. The CSL could replace yeast extract, vitamins and minerals (excluding NH₄ ⁺). The optimized CSL medium produced 120 and 50 mM of ethanol and acetate, respectively. The CSL could provide as an inexpensive source of most of the nutrients required for the syngas fermentation, and thus could improve the economics of ethanol production from biomass derived synthesis gas by C. ragsdalei.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Mixotrophic productivity of the marine diatom Phaeodactylum tricornutum cultured with soluble fractions of rye,wheat and potato

The marine microalga Phaeodactylum tricornutum was cultured semi-continuously with the soluble fractions of wheat, rye and boiled potato flours. Fifteen percent of the culture volume was renewed every 3 d. The cell productivities were 0.9×109 cells/l/d, 1.1×109 cells/l/d and 2.6×109 cells/l/d for wheat, rye and potato respectively. The productivity of the autotrophic control was 1.0×109 cell/l/d. When a soluble fraction of raw potato was added, the productivity was enhanced to 4.1×109 cells/l/d, 2.4 times higher than the autotrophic culture. The high productivity of P. tricornutum with the soluble fractions of Solanum tuberosum suggests its usefulness as a source of nutrients for the production of microalgal biomass.     

Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements

The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10 × 10⁵ cell/mL was infected, producing 1.3 × 10⁹ infectious viral particles/mL (IVP/mL).To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78 × 10⁵ cell/mL with the consequent production of 44 × 10⁹ IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.