Polyurethane foam as an inert carrier for the production of L(+)-lactic acid by Lactobacillus casei under solid-state fermentation

AIM: Production of L-lactic acid in solid-state fermentation (SSF) using polyurethane foam (PUF) as inert support moistened with cassava bagasse starch hydrolysate. METHODS AND RESULTS: PUF impregnated with cassava bagasse starch hydrolysate as major carbon source was used for the production of L-lactic acid using Lactobacillus casei in solid-state condition. The key parameters such as reducing sugar, inoculum size and nutrient mixture were optimized by statistical approach using response surface methodology. More than 95% conversion of sugars to lactic acid from 4 g reducing sugar per gram dry support was attained after 72 h when the inert substrate was moistened with 6.5 ml of nutrient solution and inoculated with 1.5 x 10(9) CFU of L. casei. While considering the lactate yield based on the solid support used, a very high yield of 3.88 g lactic acid per gram PUF was achieved. CONCLUSION: PUF acted as an excellent inert support for L. casei and provided a platform for the utilization of starchy waste hydrolysate in a lower reactor volume. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a cost effective cultivation of lactic acid bacteria for producing lactic acid from agro based waste products such as cassava bagasse. This is the first report on the exploitation of PUF as an inert support for lactate production under SSF.     

Direct fermentative production of lactic acid on cassava and other starch substrates

Lactobacillus amylovorus utilized raw corn, rice and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/L, but had lower productivities of 4.8 g/L and 4.2 g/L on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% conversion to 70% conversion (7.7 g lactic acid/L). The availability of some components of protein in corn starch is assumed to be the reason for high lactic acid production as compared to that of cassava starch.     

一株厌氧嗜热杆菌生长及发酵特性的初步研究

论文对筛选并鉴定为Thermoanaerobacterium saccharolyticum菌株的生长、底物利用情况、产物生成以及酒精耐受性进行了研究。结果表明,该菌在以甘露糖、葡萄糖和木糖为碳源时生长较好,同时能够较好的利用木聚糖和木薯淀粉;最适底物浓度为15g/L;不同的葡萄糖:木糖比例对其生长无显著影响;能耐受的培养基最高初始酒精浓度为3%(V/V)。在5g/L的木聚糖、木糖和木薯淀粉培养基中发酵60h后,产物主要有乙醇、乳酸和乙酸,乙醇产量分别为0.824、0.867和0.916g/L。     

<Emphasis Type="SmallCaps">L</Emphasis>(+)-Lactic Acid Production Using <Emphasis Type="Italic">Lactobacillus Casei</Emphasis> in Solid-State Fermentation

Lactobacillus casei was grown at 37 °C on sugarcane bagasse (5 g) soaked with cassava starch hydrolysate (final moistening volume 34 ml) containing 3 g reducing sugar in a solid-state condition. The maximum yield of l-lactic acid after various process optimisations was 2.9 g/5 g initial substrate corresponding to 97% conversion of sugar to lactic acid with initial substrate moisture of 72%.     

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.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

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.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Optimization of physiological factors for direct saccharification of cassava starch to glucose by Rhizopus oligosporus 145f

Direct saccharification of 2.64% cassava starch by Rhizopus oligosporus 145F was attempted under various cultural conditions. Maximum glucose yield of 18.0 g/L culture filtrate was obtained with an initial pH 3.8, 2% (v/v) inoculum of R. oligosporus spores, and an incubation temperature of 45 degrees C in shake flask cultures for 48 h. This concomitantly produced 2.7 g mycelia/100g cassava starch containing 20.2% true protein. The production of glucose and mycelia was accomplished with 92.8% starch saccharification having 67.9% starch to glucose conversion efficiency.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid-hydrolysed cassava starch

Raw cassava starch, having 74.94 and 0.44 g/100 g resistant starch type II and III (RS II and RS III), respectively, was autoclaved at 121 °C in water, 1, 10 or 100 mmol/L lactic acid. The formation of RS III was evaluated in relation to variable incubation temperature (−20 to 100 °C), incubation time (6–48 h) and autoclaving time (15–90 min). Negligible to low quantities of RS III (0.59–2.42 g/100 g) were formed from autoclaved starch suspended in 100 mmol/L lactic acid, whereas intermediate to high quantities (2.68–9.97 g/100 g) were formed from autoclaved starch suspended in water, 1 or 10 mmol/L lactic acid, except for treatments with water or 10 mmol/L lactic acid incubated at 100 °C for 6 h (1.74 g/100 g). Autoclaving times corresponding to maximum RS III contents were 15 and 45 min for water and 10 mmol/L lactic acid, respectively. Whereas, the RS III fractions from cassava starch suspended in water had melt transitions between 158 and 175 °C with low endothermic enthalpies (0.2–1.6 J/g), the thermal transitions of the acid treated samples were indistinct.     

Fermentation of sugar cane bagasse hemicellulose hydrolysate to l(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.**

Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to L(+)-lactic acid using a newly isolated strain of Bacillus sp. In batch fermentations at 50 degrees C and pH 5, over 5.5% (w/v) L(+)-lactic acid was produced (89% theoretical yield; 0.9 g lactate per g sugar) with an optical purity of 99.5%.     

Direct fermentation of various pure and crude starchy substrates to L(+) lactic acid using Lactobacillus amylophilus GV6

Lactobacillus amylophilus GV6 fermented a variety of pure and natural starches directly to L(+) lactic acid. Starch to lactic acid conversion efficiency was more than 90% by strain GV6 at low substrate concentrations with all starches. The strain GV6 produced high yields of lactic acid per g of substrate utilized with pure starches such as soluble starch, corn starch, and potato starch, yielding 92–96% at low substrate concentrations in 2 days and 78–89% at high substrate (10%) concentrations in 4–6 days. Strain GV6 also produced high yields of lactic acid per g of substrate utilized with crude starchy substrates such as wheat flour, sorghum flour, cassava flour, rice flour and barley flour yielding 90–93% at low substrate concentrations in 2 days and 80% or more at high substrate concentrations in 6–7 days. Lactic acid yields by L. amylophilus GV6 with pure starches were comparable when low cost crude starchy substrates were used. Lactic acid productivity by strain GV6 is higher than for any other previously reported strains of L. amylophilus.     

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.     

Use of inexpensive nitrogen sources and starch for L(+) lactic acid production in anaerobic submerged fermentation

L(+) Lactic acid fermentation was studied by Lactobacillus amylophilus GV6 under the influence of inexpensive nitrogen sources (red lentil-RL, and Baker's yeast cells-YC) and starch by response surface methodology (RSM). Central composite rotatable design (CCRD) was employed to determine maximum lactic acid production at optimum values for process variables RL, YC and incubation period (IP) and a satisfactory fit model was realized. Lactic acid production was significantly affected by RL and IP interactions as well as by independent variables RL and YC. Maximum lactic acid production of 13.5 g/15.2g starch was obtained with RL 0.8%, YC 1% and IP of 48 h, with 92% lactic acid yield efficiency (g lactic acid produced/g substrate utilized) and 40% increase (from 50 g to 92 g/100 g starch utilized) in lactic acid production. This is the first report on response optimization in direct fermentation of starch to lactic acid using inexpensive nitrogen sources substituting peptone and yeast extract in anaerobic submerged fermentation by amylolytic lactic acid bacteria (LAB).     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

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.     

Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes

Five agricultural wastes were evaluated in submerged fermentation for xylanolytic enzymes production by Penicillium janthinellum. The wastes were hydrolyzed in acid medium and the liquid fraction was used for cultivation. Corn cob (55.3 U/mL) and oat husk (54.8 U/mL) were the best inducers of xylanase. Sugar cane bagasse (23.0 U/mL) and corn husk (23.8 U/mL) were moderately good, while cassava peel was negligible. Protease production was very low in all agro-industrial residues. The maximum biomass yields were 1.30 and 1.17 g/L for cassava peel and corn husk after 180 h, respectively. Xylanolytic activity showed a cell growth associated profile.     

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.     

Lactic acid fermentation on barley flour without additional nutrients

Summary An amylolytic lactic acid producing Lactobacillus amylovorus produced 36 g/l of lactic acid in mixed cultures with L. casei without additional nutrients at 37 °C in 48 h, when barley flour concentration was 180 g/l (appr. 108 g/l starch) and barley malt quantity 0.8% of flour weight. This represented an improvement of up to 20% in comparison to the fermentation with L. amylovorus or L. casei alone. By simultaneous glucoamylase addition lactic acid production yield was about doubled. With L. casei the lactic acid yield was from 580 g in 72 h to 667 g in 144 h per kg barley flour.