Direct conversion of starch to L(+) lactic acid by amylase producing Lactobacillus amylophilus GV6

Lactobacillus amylophilus strain GV6, isolated from corn starch processing industrial wastes, was amylolytic and produced 0.96?g L(+) lactic acid per gram of soluble starch. The optimum temperature and pH for growth and L(+) lactic acid production were 37?°C and 6.5, respectively. At low substrate concentrations, the lactic acid production on corn starch was almost similar to soluble starch. The strain is fermenting various naturally available starches directly to lactic acid. The total amylase activity of the strain is 0.59?U/ml/min. The strain produced 49 and 76.2?g/l L(+) lactic acid from 60?g/l corn starch and 90?g/l soluble starch, respectively. This is the highest L(+) lactic acid among the wild strains of L. amylophilus reported so far.     

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure l-lactic acid from glucose and starch. In batch fermentation at pH?6.0, 240 g/L of glucose was completely consumed giving 210 g/L of l-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of l-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.     

Efficient Itaconic acid production via protein–protein scaffold introduction between GltA,AcnA, and CadA in recombinant Escherichia coli

Itaconic acid, which is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein–protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/L of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30°C, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/L.     

Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440

Abstract

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95?±?1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87?±?0.38% molar yield. With fermentation time of less than 24?h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Lipids-free waxy corn starch as a substrate for distillery yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Utilization of lipids-free waxy starch by distillery yeasts in fuel ethanol production can contribute to better management of renewable resources, like cereals, especially maize Zea mays L. But the efficient conversion of starch into glucose-rich fermentable substrate, and subsequently to ethanol, needs more research on hydrolysis and fermentation conditions. The aim of this study was to evaluate the lack of natural corn grain lipids on the process of simultaneous saccharification and fermentation using chemometric techniques of designed experiments, commercial enzymatic preparations and distillery yeasts Saccharomyces cerevisiae CCY-11-3. Based on the results and statistical software support we can conclude that extraction of lipids from corn grains did not lead to statistically significant increase or decrease of glucose concentration in starch hydrolysis. The ethanol concentration in fermentation mash according to analysis was not statistically significantly affected by lipids extraction. The separated lipids could serve as a source of very valuable corn oil.     

Process design and optimization of bioethanol production from cassava bagasse using statistical design and genetic algorithm

Abstract

Bioethanol production from agro-industrial residues is gaining attention because of the limited production of starch grains and sugarcane, and food–fuel conflict. The aim of the present study is to maximize the bioethanol production using cassava bagasse as a feedstock. Enzymatic liquefaction, by α-amylase, followed by simultaneous saccharification and fermentation (SSF), using glucoamylase and Zymomonas mobilis MTCC 2427, was investigated for bioethanol production from cassava bagasse. The factors influencing ethanol production process were identified and screened for significant factors using Plackett–Burman design. The significant factors (cassava bagasse concentration (10–50?g/L), concentration of α-amylase (5–25% (v/v), and temperature of fermentation (27–37?°C)) were optimized by employing Box–Behnken design and genetic algorithm. The maximum ethanol concentrations of 25.594?g/L and 25.910?g/L were obtained from Box–Behnken design and genetic algorithm, respectively, under optimum conditions. Thus, the study provides valuable insights in utilizing the cost-effective industrial residue, cassava bagasse, for the bioethanol production.     

Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10

A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20+/-2.0 and 20.0+/-1.0 g l(-1), respectively. Itaconic acid yields of 28.5+/-2.2 and 31.0+/-1.7 g l(-1) were obtained with acid and alpha-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain.     

Optimization of propionic acid production in apple pomace extract with Propionibacterium freudenreichii

Abstract

Sequential optimization of propionate production using apple pomace was studied. All experiments were performed in a static flask in anaerobic conditions. Effect of apple pomace as nitrogen source against conventional N sources (yeast extract, peptone) was studied. The double increase was observed in propionic acid production while using yeast extract and peptone (0.29?±?0.01?g/g), as against the use of only apple pomace extract (APE) (0.14?±?0.01?g/g). Intensification of propionic acid fermentation was also achieved by increasing the pH control frequency of the culture medium from 24-(0.29?±?0.01?g/g) to 12-hour intervals (30?°C) (0.30?±?0.02?g/g) and by increasing the temperature of the culture from 30 to 37?°C (12-hour intervals of pH control) (0.32?±?0.01?g/g). An important factor in improving the parameters of fermentation was the addition of biotin to the medium. The 0.2?mg/L dose of biotin allowed to attain 7.66?g/L propionate with a yield of 0.38?±?0.03?g/g (12-hour intervals of pH control, 37?°C).     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Valorization of Hemicelluloses: Production of Bioxylitol from Poplar Wood Prehydrolyzates by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> FTI 20037

In this study on the valorization of hemicelluloses (a co-product generated during cellulosic bioethanol production), prehydrolyzates obtained from poplar woodchips pretreated in an industrial experimental steam-explosion pilot-plant facility were evaluated for the production of bioxylitol using the yeast, Candida guilliermondii FTI 20037, employing both batch and fed-batch fermentation modes in shake flasks on defined nutrient medium. The prehydrolyzates consisted of monosaccharides (pentose and hexose sugars) as well as xylo-oligosaccharides and undegraded hemicellulose. Xylose (31.6?±?0.57 g/L) was the major sugar in the prehydrolyzates that also contained acetic acid and degradation products of lignin and sugars (phenolic and furanic compounds). Xylose in the prehydrolyzates could be further increased (106.4?±?0.02 g/L) through an acid hydrolysis step (0.6 % (w/v) H₂SO₄). Compounds of a toxic nature in both the acid hydrolyzates and prehydrolyzates were removed by treatment with Amberlite IRA-400 resin (chloride form). Batch fermentation of pure xylose and poplar prehydrolyzate resulted in bioxylitol production of 9.9?±?0.01 and 4.9?±?0.17 g/L, respectively, indicating that the poplar prehydrolyzates exhibited an inhibitory effect on fermentation. After detoxification of the poplar prehydrolyzates, bioxylitol production increased to 8.9?±?0.01 g/L. Fed-batch fermentation of the prehydrolyzate increased the bioxylitol production to 12.39?±?0.33 g/L, while acid hydrolysis followed by detoxification resulted in a maximum bioxylitol production of 22.0?±?0.01 g/L, a 348 % increase. The results demonstrated that acid hydrolysis and detoxification followed by fed-batch fermentation was an efficient way to produce bioxylitol from poplar prehydrolyzates.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Cyanophycin production from feather hydrolysate using biotechnological methods

Abstract

Cyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93?h with degradation rate of 96.5%. A concentration of 60?g/L of FH, generated by keratinolytic activity (8?×?10³?U?g^?1L^?1d^?1) within 24?h, was used as the main carbon/peptone source to produce cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1?±?0.4 and 5.3?±?0.3?g/L of cell mass were obtained after 72?h from FH and CA, respectively. Cyanophycin production yielded 1.4?±?0.1g/L for FH with average molecular mass of 28.8 and 1.4?±?0.2 for CA with average molecular mass of 35.3, after 60?h. For the first time, FH generated by biotechnological methods from environmentally problematic, abundant and renewable feather bioresource was successfully used for cyanophycin biopolymer production.     

Process development of itaconic acid production by a natural wild type strain of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> to reach industrially relevant final titers

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.     

Strain development and medium optimization for fumaric acid production

Rhizopus oryzae RUR709 mutant was isolated based on halo size from selection medium via mutagenesis with UV and γ-rays, and the production of fumaric acid in the submerged fermentation was assessed. The maximum concentration of fumaric acid was obtained using 0.5% corn steep liquor (CSL) as the nitrogen source. Organic nitrogen sources were shown to be more effective in fumaric acid production than inorganic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentration of fumaric acid achieved in flask culture was 26.2 g/L, which is fairly close to the 27.4 g/L predicted by the model. The highest concentration of fumaric acid in the stirred-tank reactor generated by the R. oryzae RUR709 mutant was 32.1 g/L and yield (0.45 g/g) and productivity (0.32 g/L/h) were highest at 4 days.     

Efficient bioconversion from acid hydrolysate of waste oleaginous yeast biomass after microbial oil extraction to bacterial cellulose by Komagataeibacter xylinus

Biomass acid hydrolysate of oleaginous yeast Trichosporon cutaneum after microbial oil extraction was applied as substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus (also named as Gluconacetobacter xylinus previously) for the first time. BC was synthesized in static culture for 10 days, and the maximum BC yield (2.9?g/L) was got at the 4th day of fermentation. Most carbon sources in the substrate (glucose, mannose, formic acid, acetic acid) can be utilized by K. xylinus. The highest chemical oxygen demand (COD) removal (40.7?±?3.0%) was obtained at the 6th day of fermentation, and then the COD increased possibly due to the degradation of BC. The highest BC yield on COD consumption was 38.7?±?4.0% (w/w), suggesting that this is one efficient bioconversion for BC production. The BC structure was affected little by the substrate by comparison with that generated in classical HS medium using field-emission scanning electron microscope (FE-SEM), Fourier transform infrared, and X-ray diffraction. Overall, this technology can both solve the issue of waste oleaginous yeast biomass and produce valuable biopolymer (BC).     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.