Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

Impact of balanced substrate flux on the metabolic process employing fuzzy logic during the cultivation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var.<Emphasis Type="Italic"> Galleriae</Emphasis>

Summary The insecticidal crystal protein (ICP) synthesized at the onset of sporulation by Bacillus thuringiensis var. galleriae (Btg) is lethal against specific pests. Attempts were made to enhance the synthesis of biomass and ICP by Btg employing process optimization strategies. The process optimization was carried out with residual glucose concentration control in a bench scale bioreactor. A fuzzy logic-based feedback control system for maintaining the residual glucose concentration at a constant level during cultivation was developed in LabVIEW. This control system indicated the possibilities in providing a balanced substrate flux during cultivation. The identified optimum level of 2.72 g/l in residual glucose concentration was maintained by fed-batch cultivation with glucose and yeast extract fed at equal concentration with the above control system. High cell density of 16.0 g/l with specific growth rate of 0.69 h^-1 was obtained during the cultivation. The balanced flux of substrate during cultivation has resulted in the enhanced synthesis of biomass and ICP. This optimized process could be commercially exploited by comparing the fluxes of basal compounds in different media sources used in fermentation.     

Cost-effective production of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> using simple netting bag solid bioreactor

A novel simple solid state fermentation method, netting bag bioreactor (Φ 120 × 800 mm), was developed and used to cultivate Bacillus licheniformis as probiotics. High spore yield (1.2 × 10¹¹ CFU/g dry substrate) has been obtained by using this method. Comparing to the tray bioreactor and the packed bed bioreactor for Bacillus fermentation, the netting bag method was more cost-effective, time- and space-saving and the material cost is also as low as ca. US $293 per 1,000 kg spores. Thus, netting bag SSF can be widely applied to produce probiotic bacteria in developing areas.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. HX-308

Docosahexaenoic acid (DHA) production in Schizochytrium sp. HX-308 was evaluated by detecting enzymatic activities of ATP:citrate lyase (EC 4.1.3.8), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at different fermentation stages. According to the analysis, a regulation strategy was proposed which reinforced acetyl-CoA and NADPH supply at a specific fermentation stage. DHA content of total fatty acids was increased from 35 to 60% by the addition of 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid content also showed an apparent increase of 35% and reached 19 g/L when 40 mL ethanol/L was added at the late lipid accumulation stage.     

Oxygen supply in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> fermentations: bringing new insights on their impact on sporulation and δ-endotoxin production

The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC₅₀ of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC₅₀ of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

A cost-effective medium for the large-scale production of Bacillus sphaericus H5a5b (VCRC B42) for mosquito control

Bacillus sphaericus has been widely used in mosquito control programs, but the large-scale production of this bacterium is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max) and peanut cake powder (Arachis hypogea) by using 100-l bioreactor. Sporulation, toxicity and biomass were satisfactory after B. sphaericus was produced on both media. Use of the soybean culture medium resulted in “maximum” toxicity (LC₅₀ 14.02 ng/ml against third instar Culex quinquefasciatus larvae), highest spore count (3.7 × 10⁹ spores/ml) and maximum biomass (4.6 g/l) within a short fermentation time of 21 h. Hence, this soybean-based culture medium was considered most economical for the large-scale industrial production of B. sphaericus.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

Inhibition of <Emphasis Type="Italic">Pichia stipitis</Emphasis> fermentation of hydrolysates from olive tree cuttings

The ethanolic fermentation of liquid fractions (hydrolysates) issued from dilute acid pre-treatment of olive tree biomass by Pichia stipitis is reported for the first time. On the one side, P. stipitis has been reported as the most promising naturally occurring C5 fermenting microorganism; on the other side, olive tree biomass is a renewable, low cost, and lacking of alternatives agricultural residue especially abundant in Mediterranean countries. The study was performed in two steps. First, the fermentation performance of P. stipitis was evaluated on a fermentation medium also containing the main inhibitors found in these hydrolysates (acetic acid, formic acid, and furfural), as well as glucose and xylose as carbon sources. The effect of inhibitors, individually or in a mixture, on kinetic and yield parameters was calculated. In a second step, hydrolysates obtained from 1% (w/w) sulfuric acid pre-treatment of olive tree biomass at 190°C for 10 min were used as a real fermentation medium with the same microorganism. Due to inhibition, effective fermentation required dilution of the hydrolysate and either overliming or activated charcoal treatment. Results show that ethanol yields obtained from hydrolysates, ranging from 0.35 to 0.42 g/g, are similar to those from synthetic medium, although the process proceeds at lower rates. Inhibiting compounds affect the fermentation performance in a synergistic way. Furfural is rapidly assimilated by the yeast; acetic acid and formic acid concentrations decrease slowly during the process. Activated charcoal or overliming detoxification improve the fermentability of diluted hydrolysates.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Kinetics of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> growth on high glucose concentrations

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S ₀) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S ₀ values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ _X,m) were not affected with S ₀ up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in μ _X,m values. These higher S ₀ values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X _m = 15.2–15.9 g/l) were achieved with S ₀ over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S ₀ from 30.8 to 152 g/l, cell yield (Y _X/S ) decreased from 0.58 to 0.41 g/g. On the other hand, with S ₀ = 10 g/l growth was limited by substrate, and Y _X/S has shown its maximum value (0.83 g/g).     

High cell density fermentation of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003 for glycolic acid production

Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.     

Development of a cost-effective medium for the large scale production of Bacillus thuringiensis var israelensis

Bacillus thuringiensis var israelensis (B.t.i.) has been widely used in mosquito control programs, but the large scale production of this bacillus is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max), groundnut cake powder (Arachis hypogea), and wheat bran extract (Triticum aestivum) by using 100-L fermentor. Sporulation, toxicity, and biomass were satisfactory after B.t.i. was produced on all the three media. Use of the soybean culture medium resulted in maximum toxicity (LC₅₀ 8.89 ng/ml against Culex quinquefasciatus IIIrd instar larvae), highest spore count (0.48 × 10¹¹ c.f.u./ml), and maximum biomass (7.8 g/L) within a short fermentation time of 24 h. Hence, this soybean-based culture medium was considered most economical for the large scale industrial production of B.t.i.     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.