Improved enzymatic saccharification of steam exploded cotton stalk using alkaline extraction and fermentation of cellulosic sugars into ethanol.

Cotton stalk, a widely available and cheap agricultural residue lacking economic alternatives, was subjected to steam explosion in the range 170-200°C for 5min. Steam explosion at 200°C and 5min led to significant hemicellulose solubilization (71.90±0.10%). Alkaline extraction of steam exploded cotton stalk (SECOH) using 3% NaOH at room temperature for 6h led to 85.07±1.43% lignin removal with complete hemicellulose solubilization. Besides, this combined pretreatment allowed a high recovery of the cellulosic fraction from the biomass. Enzymatic saccharification was studied between steam exploded cotton stalk (SECS) and SECOH using different cellulase loadings. SECOH gave a maximum of 785.30±8.28mg/g reducing sugars with saccharification efficiency of 82.13±0.72%. Subsequently, fermentation of SECOH hydrolysate containing sugars (68.20±1.16g/L) with Saccharomyces cerevisiae produced 23.17±0.84g/L ethanol with 0.44g/g yield.

Enhanced rifamycin SV production by submerged fermentation using Amycolatopsis mediterranei.

Rifamycin is a broad-spectrum antimicrobial drug produced commercially by submerged fermentation where the yields are far less in comparison to its demand in human drug therapy. Addressing the need, sequential mutational strain improvement was carried using UV and EtBr that resulted in improved strain yielding rifamycin SV up to 4.32 g/L. Further optimization of six important fermentation factors was followed which include temperature, agitation, inoculum level, period of fermentation, inorganic nitrogen source and amino acids. For the first time, we report a maximum yield of 5.32 g/L of rifamycin SV. Among the amino acids, proline known for its slowest assimilation by Amycolatopsis mediterranei produced the highest improvement in antibiotic yields. Following mutational strain improvement and process optimization, a total of 3.8-fold increase in antibiotic titre was achieved. Following a conventional procedure of mutational strain improvement, highest yield of rifamycin SV was reported by optimizing submerged fermentation process.

Direct bioethanol production by amylolytic yeast Candida albicans.

UNLABELLED: An attempt was made to produce bioethanol using optimized fermentation parameters and mutationally improved strain of Candida albicans. The mutant strain OMC3E6 obtained by UV irradiation followed by ethidium bromide successive mutations showed 2.6 times more glucoamylase secretion and 1.5 times more bioethanol production via direct conversion of starch. Enhanced hydrolysis of insoluble starch (72%) and potato starch (70%) was achieved with glucoamylase enzyme preparation from mutant C. albicans. In fermentation medium, the use of maltose, corn steep liquor, NaH2 PO4 , NaCl + MgSO4 and Triton X-100 has increased the glucoamylase production by the microbe. Under optimized conditions, C. albicans eventually produced 437 g ethanol kg(-1) potatoes. Earlier reports mentioned the use of thrice the quantity of starch as reported by us followed by more fermentation period (3-4 days) and demanded pretreatment of starch sources with alpha-amylase as well. Here, we simplified these three steps and obtained 73% conversion of insoluble starch into ethanol via direct conversion method in a period of 2 days without the involvement of cell immobilizations or enzyme pretreatment steps. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to fast depletion of fossil fuels in the modern world, bioethanol usage as an alternate energy source is the need of the hour. For the first time, we report bioethanol production by Candida albicans via direct conversion of starchy biomass into ethanol along with enhanced starch-hydrolysing capacity and ethanol conversion ratio. So far, C. albicans was dealt in the field of clinical pathology, but here we successfully employed this organism to produce bioethanol from starchy agri-substrates. Optimizing fermentation parameters and improving the microbial strains through successive mutagenesis can improve the end product yield.

Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis.

Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses). Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L(-1). However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L(-1) by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L(-1), with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L(-1)h(-1), 0.4 g g(-1) and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498.

Bioconversion of novel substrate Saccharum spontaneum, a weedy material, into ethanol by Pichia stipitis NCIM3498.

Rising fuel prices and environmental issues have paved the way for the exploration of cellulosic ethanol. However, challenges involving substrate hydrolysis and cost-effectiveness still limit the efficient bioconversion and utilization of cellulosic ethanol. We aimed to evaluate a cheaper and abundantly available wild sugarcane variety, Saccharum spontaneum, as the raw substrate for bioconversion of ethanol by Pichia stipitis NCIM3498. Three different strategies for substrate hydrolysis using acid (dilute sulfuric acid) and alkali (dilute sodium hydroxide) and aqueous ammonia (AA) treatment followed by enzymatic hydrolysis were studied. A maximum of 631.5±3.25 mg/g sugars with 89.38% hydrolytic efficiency (HE) could be achieved after enzymatic hydrolysis of AA-pretreated S. spontaneum. All the substrate hydrolysates were evaluated for ethanol conversion in batches by P. stipitis. The microbial fermentation of released sugars into ethanol showed (g/g) 0.36±0.011, 0.384±0.022, 0.391±0.02, and 0.40±0.01 yield from detoxified acid hydrolysate and acid-, NaOH- and AA-pretreated substrate S. spontaneum enzymatic hydrolysates, respectively.

Bioconversion of Saccharum spontaneum (wild sugarcane) hemicellulosic hydrolysate into ethanol by mono and co-cultures of Pichia stipitis NCIM3498 and thermotolerant Saccharomyces cerevisiae-VS3.

The lignocellulosic biomass is a low-cost renewable resource for eco-benign liquid fuel 'ethanol'. To resolve the hydrolysis of mixed sugars in lignocellulosic substrate Saccharum spontaneum, the microbial co-cultures of Pichia stipitis NCIM 3498 and thermotolerant Saccharomyces cerevisiae-VS(3) were analyzed for efficient bioconversion of mixed sugars into ethanol. Among the hydrolysis conditions, the acid hydrolysis released maximum sugars along with furans, phenolics and acetic acid. The acidic hydrolysate was detoxified and fermented by monocultures of P. stipitis NCIM3498 (P.S.) and thermotolerant S. cerevisiae VS(3) (S.C.), and co-culture of P.S. (7.5 mL) and S.C. (2.5 mL). Before the fermentation of hemicellulose acid hydrolysate, both the monocultures (P.S. and S.C.), and varying ratios of P.S. and S.C. microorganisms in co-cultures #1, #2 and #3 were grown on simulated synthetic medium. The ethanol yield from monocultures of P.S. (0.44 ± 0.021 g/g), S.C. (0.22 ± 0.01 g/g) and co-culture #3 (0.49 ± 0.02 g/g) revealed unique characteristics of each mono and co-culture technology. The fermentation of hemicellulose acid hydrolysate with monocultures of P.S., S.C. and co-culture #3 produced 12.08 ± 0.72 g/L, 1.40 ± 0.07 g/L, and 15.0 ± -0.92 g/L ethanol, respectively.

Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3.

Saccharum spontaneum is a wasteland weed consists of 45.10+/-0.35% cellulose and 22.75+/-0.28% of hemicellulose on dry solid (DS) basis. Aqueous ammonia delignified S. spontaneum yielded total reducing sugars, 53.91+/-0.44 g/L (539.10+/-0.55 mg/g of substrate) with a hydrolytic efficiency of 77.85+/-0.45%. The enzymes required for hydrolysis were prepared from culture supernatants of Aspergillus oryzae MTCC 1846. A maximum of 0.85+/-0.07 IU/mL of filter paperase (FPase), 1.25+/-0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56+/-0.52 IU/mL of xylanase activity was obtained after 7 days of incubation at 28+/-0.5 degrees C using delignified S. spontaneum as carbon source under submerged fermentation conditions. Enzymatic hydrolysate of S. spontaneum was then tested for ethanol production under batch and repeated batch production system using "in-situ" entrapped Saccharomyces cerevisiae VS3 cells in S. spontaneum stalks (1 cm x 1 cm) size. Immobilization was confirmed by the scanning electron microscopy (SEM). Batch fermentation of VS3 free cells and immobilized cells showed ethanol production, 19.45+/-0.55 g/L (yield, 0.410+/-0.010 g/g) and 21.66+/-0.62 g/L (yield, 0.434+/-0.021 g/g), respectively. Immobilized VS3 cells showed maximum ethanol production (22.85+/-0.44 g/L, yield, 0.45+/-0.04 g/g) up to 8th cycle during repeated batch fermentation followed by a gradual reduction in subsequent cycles of fermentation.

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates.

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.

Lantana camara for fuel ethanol production using thermotolerant yeast.

AIM: Evaluation of Lantana camara's use as feedstock for fuel ethanol production. METHODS AND RESULTS: Lantana camara plant material was hydrolysed with 1% sulfuric acid for 18 h at room temperature, followed by heat treatment of 121 degrees C for 20 min. Hemicellulosic hydrolyzate was separated and used for detoxification by ethyl acetate and overliming. Cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 55 degrees C. Using 15% (dw/v) substrate 73 g l(-1) total reducing sugars were obtained to give 78.7% hydrolysis of carbohydrate content. Acid and enzyme hydrolyzates were mixed equally and used for fermentation with thermotolerant Saccharomyces cerevisiae (VS(3)). Yeast fermented L. camara hydrolyzate well with a fermentation efficiency of 83.7% to give an ethanol yield of 0.431 +/- 0.018 g ethanol pre g sugar and productivity of 0.5 +/- 0.021 g l(-1) h(-1). CONCLUSIONS: Even though inhibitors were present in L. camara hydrolyzate, maximum sugars were utilized by thermotolerant yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of L. camara for fuel ethanol production with improved strains and detoxification can be recommended.

Xylitol production from corn fiber and sugarcane bagasse hydrolysates by Candida tropicalis.

A natural isolate, Candida tropicalis was tested for xylitol production from corn fiber and sugarcane bagasse hydrolysates. Fermentation of corn fiber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production, though these were very low, even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was found to be 0.43 g/g and 0.45 g/g of xylose utilised with corn fiber and sugarcane bagasse hydrolysate respectively. One of the critical factors for low xylitol production was the presence of inhibitors in these hydrolysates. To simulate influence of hemicellulosic sugar composition on xylitol yield, three different combinations of mixed sugar control experiments, without the presence of any inhibitors, have been performed and the strain produced 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. To improve yeast growth and xylitol production with these hydrolysates, which contain inhibitors, the cells were adapted by sub culturing in the hydrolysate containing medium for 25 cycles. After adaptation the organism produced more xylitol 0.58 g/g and 0.65 g/g of xylose with corn fiber hydrolysate and sugarcane bagasse hydrolysate respectively.

Antimicrobial activities of Eugenol and Cinnamaldehyde against the human gastric pathogen Helicobacter pylori.

BACKGROUND: Eradication of Helicobacter pylori is an important objective in overcoming gastric diseases. Many regimens are currently available but none of them could achieve 100% success in eradication. Eugenol and cinnamaldehyde that are commonly used in various food preparations are known to possess antimicrobial activity against a wide spectrum of bacteria. AIM: The present study was performed to assess the in vitro effects of eugenol and cinnamaldehyde against indigenous and standard H. pylori strains, their minimum inhibitory concentrations (MICs) and time course lethal effects at various pH. METHODS: A total of 31 strains (29 indigenous and one standard strain of H. pylori ATCC 26695, one strain of E. coli NCIM 2089) were screened. Agar dilution method was used for the determination of drug sensitivity patterns of isolates to the commonly used antibiotics and broth dilution method for the test compounds. RESULTS: Eugenol and cinnamaldehyde inhibited the growth of all the 30 H. pylori strains tested, at a concentration of 2 mug/ml, in the 9th and 12th hours of incubation respectively. At acidic pH, increased activity was observed for both the compounds. Furthermore, the organism did not develop any resistance towards these compounds even after 10 passages grown at sub-inhibitory concentrations. CONCLUSION: These results indicate that the two bioactive compounds we tested may prevent H. pylori growth in vitro, without acquiring any resistance.

Association between cag-pathogenicity island in Helicobacter pylori isolates from peptic ulcer, gastric carcinoma, and non-ulcer dyspepsia subjects with histological changes.

AIM: To investigate the presence of the cag-pathogenicity island and the associated histological damage caused by strains with complete cag-PAI and with partial deletions in correlation to the disease status. METHODS: We analyzed the complete cag-PAI of 174 representative Helicobacter pylori (H pylori ) clinical isolates obtained from patients with duodenal ulcer, gastric ulcer, gastric cancer, and non-ulcer dyspepsia using eight different oligonucleotide primers viz cagA1, cagA2, cagAP1, cagAP2, cagE, cagT, LEC-1, LEC-2 spanning five different loci of the whole cag-PAI by polymerase chain reaction (PCR). RESULTS: The complete screening of the genes comprising the cag-PAI showed that larger proportions of subjects with gastric ulcer (97.8%) inhabited strains with complete cag-PAI, followed by gastric cancer (85.7%), non-ulcer dyspepsia (7.1%), and duodenal ulcer (6.9%), significant differences were found in the percentage distribution of the genes in all the clinical groups studied. It was found that strains with complete cag-PAI were able to cause severe histological damage than with the partially deleted ones. CONCLUSION: The cag-PAI is a strong virulent marker in the disease pathogenesis as it is shown that a large number of those infected with strain with complete cag-PAI had one or the other of the irreversible gastric pathologies and interestingly 18.5% of them developed gastric carcinoma. The presence of an intact cag-PAI correlates with the development of more severe pathology, and such strains were found more frequently in patients with severe gastroduodenal disease. Partial deletions of the cag-PAI appear to be sufficient to render the organism less pathogenic.

Effect of UV radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains.

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.