Xylitol conversion by fermentation using five yeast strains and polyelectrolyte-assisted ultrafiltration

Batch fermentations for xylitol production were conducted using Candida boidinii (BCRC 21432), C. guilliermondii (BCRC 21549), C. tropicalis (BCRC 20520), C. utilis (BCRC 20334), and P. anomala (BCRC 21359) together with a mixture of sugars simulating lignocellulosic hydrolysates as the carbon source. C. tropicalis had the highest bioconversion yield (Y_P/S) of 0.79 g g⁻¹ (g xylitol·g xylose⁻¹) over 48 h. Additional fermentations with C. tropicalis achieved Y_P/S values of 0.6 and 0.39 g g⁻¹ after 96 and 72 h using urea and soybean meal as the nitrogen sources, respectively. Ethanol and arabitol were also produced in all fermentation. Xylitol in the fermentation broth was recovered by cross-flow ultrafiltration. With prior application of 2 mg polydiallyl dimethylammonium chloride l⁻¹ on the membrane surface, protein in the permeate was reduced from 7.1 to 1.5 mg l⁻¹ after 2 h.     

Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 μm and 50 μm As₂O₃) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Production of raw-starch-hydrolysing α-amylase from the newly isolated Geobacillus thermodenitrificans HRO10

An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 2⁴ and 3² factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T² − 8.448pH² + 6.020TpH − 0.005T²pH²) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important observation.     

Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK _m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.     

Fungal pretreatment to enhance the yield of phytochemicals and evaluation of α-amylase and α-glucosidase inhibition using Cinnamomum zeylanicum (L.) quills pressurized water extracts

Bioactive compounds entrapped in plant materials can be effectively recovered using fungal enzymes. Cinnamomum zeylanicum Sri Wijaya (SW) and Sri Gemunu (SG) accessions and commercially available C. zeylanicum (CC) were subjected to fungal pretreatment and extracted with pressured water (PWE, 0·098 MPa). Thirteen fungal species were isolated and the substrate utilization ability of the species was tested using cellulose, pectin and lignin (indirectly). Total phenolic content (TPC, Folin–Ciocalteu method), proanthocyanidin content (PC, vanillin method) and α-amylase and α-glucosidase inhibitory potential of the extracts were evaluated. The anti-diabetic drug, Acarbose was used as the positive control. Trichoderma harzianum (MH298760) showed the highest cell lysis ability and hence was used for the microbial pretreatment process. Extracts of SW treated with T. harzianum species (Pre-SW) gave the highest percentage yield (4·08% ± 0·15%), significantly potent inhibition (P < 0·05) of α-amylase and α-glucosidase activities (IC₅₀ 57 ± 8 and 36 ± 8 μg ml⁻¹ respectively), TPC (2·24 ± 0·02 mg gallic acid equivalent g⁻¹), and PC (48·2 ± 0·4 mg of catechin equivalent g⁻¹) compared to Pre-SG, Pre-CC and nontreated samples. Trichoderma harzianum treatment can enhance the hypoglycaemic properties, PC and TPC of Cinnamon extracts and provide new insights into the recovery of phytochemicals.     

Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens

D(–)-Lactic acid was produced from cellulose by simultaneous saccharification and fermentation (SSF) in media containing cellulolytic enzymes and Lactobacillus coryniformis subsp. torquens ATCC 25600 at 39 °C and pH 5.4, yielding 0.89 g D(–)-lactic acid g^–1 cellulose at a mean volumetric productivity of 0.5 g l^–1 h^–1. No L(+)-lactic acid was found in the medium.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: occurrence in the air and clinical significance

Trichoderma harzianum, a filamentous fungus, is being widely used as a potential biopesticide. The potential of this fungus in causing skin sensitization, however, was poorly investigated as yet. The objective of this study was to monitor the occurrence of T. harzianum in the air and to explore its skin sensitizing potential. Seasonal periodicity of T. harzianum was studied for the years 2002–2004 by an Andersen air sampler. The skin sensitizing potential of T. harzianum extract was studied in 389 patients with suspected respiratory allergy by skin prick test (SPT) and specific IgE level was determined by ELISA. SDS–PAGE and immunoblotting were also performed. T. harzianum colony count varied from 3.69 to 134.88 CFU m⁻³ with the peak achieved in February. Relative humidity was found to be a significant (P < 0.05) factor predicting the occurrence of T. harzianum in the air. Positive skin reaction (wheal diameter ≥ 3 mm) was observed in 105 patients (26.99%). T. harzianum crude extract was resolved in 18 protein bands (12–72 kDa) on SDS–PAGE (12% gel) including two IgE-binding protein bands (21 and 32 kDa). T. harzianum can be considered an important inhalant allergen.     

丁氟螨酯对二斑叶螨生长发育的影响

[目的]丁氟螨酯是一种新型酰基乙腈类非内吸性杀螨剂,对害螨的各个螨态都有很高活性,具有较高的应用价值.本文评价了丁氟螨酯对二斑叶螨生长发育的影响,以期为合理用药和二斑叶螨的综合防治提供理论依据.[方法]采用浸叶法测定丁氟螨酯对二斑叶螨成螨与卵的致死中浓度、雌成螨产卵量、各螨态存活率以及各发育历期的影响.[结果]经丁氟螨...     

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L^?1 for rice straw, wheat bran, peptone, and CaCO₃, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL^?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.     

Effects of Temperature and Body Size on the Swimming Speed of Larval and Juvenile Atlantic Cod (Gadus Morhua): Implications for Individual-based Modelling

Synopsis The routine swimming speed (S) of three groups of 4, 9 and 32 cm total length (L_T) juvenile cod (Gadus morhua) was quantified in the laboratory at 6 – 10 different temperatures (T) between 3.2 and 16.7°C. At temperatures between 5 and 15°C, mean group S increased exponentially with increasing T (S=a e^bT) and the effect of temperature (b = 0.082, Q₁₀ = 2.27) was not significantly different among the groups (over the 8-fold difference in fish sizes of early- and post-settlement juveniles). Differences in mean S among individuals within each group were quite large (coefficient of variation = 40 – 80%). Swimming data for juveniles and those collected for groups of 0.4, 0.7 and 0.9 cm standard length (L_S) larvae were combined to assess the effect of body size on S. At 8°C, S (mm s⁻¹) increased with L_S (mm) according to: S = 0.26L_S^Φ−5.28L_S⁻¹, where Φ = 1.55L_S^−0.08. Relative S (body lengths s⁻¹) was related to L_S by a dome-shaped relationship having a maximum value (0.49 body lengths s⁻¹) at 18.5 – 19 mm L_S corresponding to the sizes of fish at the end of larval-juvenile metamorphosis. Previous larval cod IBM’s using a cruise-predator mode likely overestimated rates of foraging (prey searching and encounters) by a factor of ~2, whereas foraging rates in pause-travel models are closer to estimates of swimming velocities obtained in this and other laboratory studies.     

Physico-chemical Approach of the Amylolytic Action Pattern of a Thermostable Amylopullulanase

This paper describes the amylolytic action pattern of Thermococcus hydrothermalis recombinant amylopullulanase (Th-ApuΔ2) [E.C 3.2.1.41]. A comparison was made between amylose hydrolysis catalyzed by this enzyme and by two other amylolytic enzymes: α-amylase [E.C 3.2.1.1] (from Aspergillus oryzae) and glucoamylase [E.C. 3.2.1.3] (from Aspergillus niger), respectively taken as models for “endo” and “exo” catalytic patterns. Different independent physico-chemical methods were used to characterize the hydrolysis products obtained with the studied enzymes. Viscosity results were correlated to reducing sugars analysis to show a similarity between glucoamylase [E.C. 3.2.1.3] and Th-ApuΔ2 [E.C 3.2.1.41] behavior. On the other hand, whereas α-amylase [E.C 3.2.1.1] action rapidly decreased the viscosity of medium, glucoamylase and Th-ApuΔ2 hydrolysates have only shown a negligible reduction in viscosity. Glass transition temperatures of glucoamylase and Th-ApuΔ2 hydrolysates were found comparable (225–226°C) and significantly different from that of α-amylase (197°C). Thin-layer chromatographic analysis of hydrolysates mainly revealed the presence of glucose in the case of glucoamylase and Th-ApuΔ2 activities and in addition to glucose the Th-ApuΔ2 chromatograms have shown oligosaccharides with polymerization degree ranging from 2 to 7. These results incite us to conclude that Th-ApuΔ2 has a dual “endo” and “exo” catalytic action pattern. Analysis of the Fourier transform infrared (FTIR) results shows a comparable general aspect for all spectra. The presence of more numerous differentiated and intense peaks in the spectrum of Th-ApuΔ2 hydrolysate reveals the presence of short-chain oligosaccharides. These results confirm thin-layer chromatography results and support a dual action pattern.     

Callus induction and plant regeneration of U.S. rice genotypes as affected by medium constituents

Summary This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L⁻¹ (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L⁻¹) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L⁻¹ (9.29 μM) kinetin combined with 0 or 0.1 mg·L⁻¹ (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.     

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5–5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The K_m values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml⁻¹, 0.65 mg ml⁻¹, and 22.64 mg ml⁻¹, respectively, and V_max values were 0.82 mol min⁻¹ mg⁻¹, 0.62 mol min⁻¹ mg⁻¹, and 104.17 mol min⁻¹ mg⁻¹ to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g⁻¹ dry substrate.

     

Micropropagation studies inCeropegia SPP

Summary The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l⁻¹) N⁶-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l⁻¹) BA and 23.2 μM (5 mg·l⁻¹) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l⁻¹) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l⁻¹) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA. Rooting of the shoots was possible both byin vitro andex vitro means.     

Production and Starch Saccharification by a Thermostable and Neutral Glucoamylase of a Thermophilic Mould Thermomucor Indicae-Seudaticae

The present investigation was aimed at producing a thermostable and neutral glucoamylase (amyloglucosidase, EC 3.2.1.3) by a thermophilic mould, Thermomucor indicae-seudaticae in submerged cultivation and testing its applicability in starch saccharification. Parametric optimization resulted in the secretion of 30,000 U/l of glucoamylase in a synthetic medium (5% soluble starch, 0.1% yeast extract, 0.05% K₂HPO₄ and 0.01% MgSO₄· 7H₂O) using 5 × 10⁶ spores/50 ml of a 3-day-old inoculum at 40 °C and 250 rev/min in shake flasks in 48 h. The enzyme secretion was not affected to any significant extent by the tested additives and detergents. A 1.7-fold increase in glucoamylase secretion was attained when T. indicae-seudaticae was grown in a laboratory fermenter. The enzyme alone catalysed the hydrolysis of soluble starch to an extent of 65%. A prior treatment of starch with thermostable α-amylase and amylopullulanase, followed by glucoamylase, resulted in a greater extent of hydrolysis, 79 and 91%, respectively.     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.