Enhancing production of prodigiosin from Serratia marcescens C3 by statistical experimental design and porous carrier addition strategy

Serratia marcescens C3 produces a natural red-pigment, prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of prodigiosin, yielding a prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO₄·4H₂O and MnSO₄·4H₂O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.     

l-Lactic acid production from liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and optimization

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing l-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH₄)₂SO_4, KH₂PO₄, MgSO₄ and ZnSO₄·7H₂O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 °C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for l-lactic acid production from low-cost starchy substrates.     

Microbial production of poly-β-hydroxybutyrate by marine microbes isolated from various marine environments

Considering the industrial interest of Poly-β-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1 g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and ¹H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Optimization of the medium for glutathione production in Saccharomyces cerevisiae

As a powerful statistical experimental design, uniform design (UD) method has been successfully applied in various fields such as fermentation industry, pharmaceuticals, and others. In this paper, UD was applied to optimize the medium composition for glutathione production in shake-flask culture of Saccharomyces cerevisiae T65. The experiments of nine factors (glucose, yeast extract, peptone, malt extract, molasses, MgSO₄, ZnSO₄, (NH₄)₂HPO₄ and thiamine) and nine levels were carried out according to the uniform design table U₂₇(9⁹). The experimental data was analyzed to obtain the regression model and the optimal medium composition was achieved by optimization with UD 3.0 software. The optimal medium consisted of 70 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, 70 g/L malt extract, 20 g/L molasses, 5.6 g/L MgSO₄, 16 mg/L ZnSO₄, 7 g/L (NH₄)₂HPO₄ and 0.2 mg/L thiamine. The GSH yield at the optimal point achieved 74.6 mg/L, which was 1.81 times higher than that of the control. The application of UD method resulted in enhancement in GSH production.     

Microbial biodegradable plastic production from a wheat-based biorefining strategy

Restructuring the current fermentation and recovery practices employed for the production of polyhydroxyalkanoates is essential for the commercialisation of environmentally benign and cost competitive biodegradable plastics. This study presents the potential of a wheat-based biorefinery for the production of poly(3-hydroxybutyrate) (PHB). Fed-batch bioconversions using Wautersia eutropha growing on wheat-derived media led to the production of 162.8 g/l PHB. A high PHB to total dry weight (TDW) yield of 93% (w/w) was achieved due to microbial autolysis at the end of fermentation. Images of bacterial cells taken with a Transmission Electron Microscope (TEM) indicated the potential of bacterial autolysis as a mean to shorten downstream processing for PHB purification. The consumption of amino acids and peptides derived from wheat gluten hydrolysis resulted in a high glucose to PHB conversion yield of 0.47 g/g. The respective yield regarding the amount of wheat used for the production of enzymes and PHB was around 0.3 g PHB/g wheat, which corresponds to 82.8% of the maximum theoretical conversion yield. The productivity achieved was around 0.9 g/l h. Fermentations carried out on wheat-derived media and media formulated with various commercial sources of nutrients (glucose, yeast extract, soy-protein acid hydrolysate, casein hydrolysates, corn steep liquor and various inorganic chemicals) showed that the proposed wheat-based biorefinery strategy enhanced PHB production.     

Astaxanthin from Jerusalem artichoke: Production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics

Jerusalem artichoke extract or powder was used for astaxanthin production using Phaffia rhodozyma without acidic or enzymatic inulin hydrolysis. The culture medium containing Jerusalem artichoke as carbon source was optimized, and feeding strategies, including constant, exponential, pH-stat, and substrate feedback fed-batch fermentations, were also compared for enhancing the cell biomass and astaxanthin synthesis by P. rhodozyma. Substrate-feedback fed-batch fermentation resulted in the highest dry cell weight of 83.60 g/L, with a carotenoid concentration and yield of 982.50 mg/L and 13.30 mg/g, respectively, under optimized medium components using Jerusalem artichoke extract as carbon source in a 3-L stirred-tank bioreactor. Moreover, 482.50 mg/L of carotenoids and 253.10 mg/L of astaxanthin were obtained by continuous feeding of Jerusalem artichoke powder, which was used as carbon source. Astaxanthin essence with high DPPH-scavenging activity was obtained from the extracted astaxanthin, and the DPPH free radical scavenging rate of 40 ppm astaxanthin essence reached 76.29%. When stored at 4 °C, astaxanthin essence showed the highest stability, with a minimum k value of 0.0099 week⁻¹ and maximum half-life (t_1/2) value of 70 weeks.     

Optimizing l-(+)-lactic acid production by thermophile Lactobacillus plantarum As.1.3 using alternative nitrogen sources with response surface method

The effects of five alternative nitrogen sources, namely, malt sprout (MS), corn steep liquor (CSL), NH₄Cl, NH₄NO₃ and diamine citrate (DC) were investigated on the l-(+)-lactic acid (LA) production by thermophile Lactobacillus plantarum As.1.3. Through the statistical analysis of the results by three steps of response surface methodology (RSM) design, MS and CSL were found to have significant effects on the LA production and their optimal concentrations in the medium should be 16.0 g/L and 12.0 g/L, respectively. The verification of the optimized medium showed that the maximum specific growth rate (μ_m) was 1.09 h⁻¹, the cell yield coefficient (Y_X/S) and the l-(+)-lactic acid yield coefficient (Y_P/S) were 0.233 (OD₆₂₀/g) and 0.98 (g/g), and the maximum volumetric productivity and the average volumetric productivity were 13.0 g/L h and 3.20 g/L h, respectively. The results indicate that the LA production can also be enhanced with the inexpensive nitrogen source alternatives.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.     

Medium optimization for the high yield production of single (+)-terrein by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca

Terrein has potential application in the fields of medicine, cosmetology and agriculture, however, the chemical synthesis of terrein with single configuration is a difficult task, and the biosynthesis of terrein always results in low production (ca. 0.33–400 mg/L). In this study, we reported an Aspergillus terreus strain PF26 which could produce (+)-terrein on a high level. After the selection of a suitable basic medium, the component concentrations were optimized using Plackett–Burman design and response surface methodology. Consequently, an optimal medium containing 28.41 g glucose, 23.18 g maltose, 20.00 g mannitol, 8.52 g malt extract, 10.00 g monosodium glutamate 10.00 g NH₄Cl in 1 L ASW was obtained, and a high (+)-terrein production of 3.71 g/L fermentation broth was achieved, which represents the highest fermentation production of (+)-terrein to date. The result highlighted the industry's potential of A. terreus strain PF26 in the production of bioactive (+)-terrein on a large-scale.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation

Crude glycerol is the primary by-product in the biodiesel industry, which is too costly to be purified into to higher quality products used in the health and cosmetics industries. This work investigated the potential of using the crude glycerol to produce docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the microalga Schizochytrium limacinum. The results showed that crude glycerol supported alga growth and DHA production, with 75–100 g/L concentration being the optimal range. Among other medium and environmental factors influencing DHA production, temperature, trace metal (PI) solution concentration, ammonium acetate, and NH₄Cl had significant effects (P < 0.1). Their optimal values were determined 30 mL/L of PI, 0.04 g/L of NH₄Cl, 1.0 g/L of ammonium acetate, and 19.2 °C. A highest DHA yield of 4.91 g/L with 22.1 g/L cell dry weight was obtained. The results suggested that biodiesel-derived crude glycerol is a promising feedstock for production of DHA from heterotrophic algal culture.     

Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO₃ combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO₃, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G⁺, glucose in the Hungate's medium as a glucose control; G^?, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract + tryptone + (NH₄)₂SO₄ in Hungate's medium (control); N2: yeast extract + (NH₄)₂SO₄; N3: tryptone + (NH₄)₂SO₄; and N4: tryptone + yeast extract). In the G^? media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G⁺ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G^? medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r > 0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.     

Enhanced production of insect raw-starch-digesting alpha-amylase accompanied by high erythritol synthesis in recombinant Yarrowia lipolytica fed-batch cultures at high-cell-densities

Recently we reported on raw-starch-digesting ability of alpha-amylase from an insect Sitophilus oryzae (SoAMY) expressed in recombinant Yarrowia lipolytica cells, and demonstrated its usefulness in simultaneous saccharification and fermentation processes with industrial yeasts. In this study we applied fed-batch cultures of Y. lipolytica 4.29 strain reaching high-cell-densities (up to 70 [g_DCW/L]), to enhance SoAMY production. SoAMY activity in the medium reached the peak value of 22,979.23 ± 184 [AU/L], at volumetric productivity of 121.58 ± 1.75 [AU/L/h], and yield of 71.83 ± 3.08 [AU/g_glycerol], constituting roughly 160-fold improvement, compared to the best previous result. The cultivations were accompanied by high production of erythritol (83.58 [g/L]), at the marginal production of mannitol (5.46 [g/L]). Elementary analyses of media constituents, the enzyme and the yeast biomass gave better insight into carbon and nitrogen fluxes distribution. Due to application of genetic engineering and bioprocess engineering strategies, the insect-derived enzyme can be produced at the quantities competitive to microbial catalysts.