Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae and Zymomonas mobilis

Mahula (Madhuca latifolia L.) flower is a suitable alternative cheaper carbohydrate source for production of bio-ethanol. Recent production of bio-ethanol by microbial fermentation as an alternative energy source has renewed research interest because of the increase in the fuel price. Saccharomyces cerevisiae (yeast) and Zymomonas mobilis (bacteria) are two most widely used microorganisms for ethanol production. In this study, experiments were carried out to compare the potential of the yeast S. cerevisiae (CTCRI strain) with the bacterium Z. mobilis (MTCC 92) for ethanol fermentation from mahula flowers. The ethanol production after 96 h fermentation was 149 and 122.9 g kg⁻¹ flowers using free cells of S. cerevisiae and Z. mobilis, respectively. The S. cerevisiae strain showed 21.2% more final ethanol production in comparison to Z. mobilis. Ethanol yield (Yx/s), volumetric product productivity (Qp), sugar to ethanol conversion rate (%) and microbial biomass concentration (X) obtained by S. cerevisiae were found to be 5.2%, 21.1%, 5.27% and 134% higher than Z. mobilis, respectively after 96 h of fermentation.     

Bioethanol production from mahula (Madhuca latifolia L.) flowers by solid-state fermentation

There is a growing interest worldwide to find out new and cheap carbohydrate sources for production of bioethanol. In this context, the production of ethanol from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae in solid-state fermentation was investigated. The moisture level of 70%, pH of 6.0 and temperature of 30 °C were found optimum for maximum ethanol concentration (225.0 ± 4.0 g/kg flower) obtained from mahula flowers after 72 h of fermentation. Concomitant with highest ethanol concentration, the maximum ethanol productivity (3.13 g/kg flower/h), yeast biomass (18.5 × 10⁸ CFU/g flower), the ethanol yield (58.44 g/100 g sugar consumed) and the fermentation efficiency (77.1%) were also obtained at these parametric levels.     

Ethanol production from mahula (Madhuca latifolia L.) flowers with immobilized cells of Saccharomyces cerevisiae in Luffa cylindrica L. sponge discs

The dried spongy fruit of luffa (Luffa cylindrica L.), a cucurbitaceous crop available in abundance in tropical and sub-tropical countries has been found to be a promising material for immobilizing microbial cells. The aim of the present study was to examine the ethanol production from mahula flowers in submerged fermentation using whole cells of Saccharomyces cerevisiae immobilized in luffa sponge discs. The cells not only survived but also were physiologically active in three more cycles of fermentation without significant reduction (<5%) in ethanol production. After 96 h, there was 91.1% sugar conversion producing 223.2 g ethanol/kg flowers (1st cycle) which was 0.99%, 2.3% and 3.2% more than 2nd (221 g ethanol/kg flowers), 3rd (218 g ethanol/kg flowers) and 4th (216 g ethanol/kg flowers) cycle of fermentation, respectively. Furthermore, ethanol production by immobilized cells was 8.96% higher than the free cells.     

Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae

To investigate the bioethanol production from sweet potato, the saccharification and fermentation conditions of co-immobilization of saccharolytic molds (Aspergillus oryzae and Monascus purpureus) with Saccharomyces cerevisiae were analyzed. The immobilized yeast cells showed that at 10% glucose YPD (yeast extract peptone dextrose) the maximum fermentation rate was 80.23%. Viability of yeasts cells were 95.70% at a final ethanol concentration of 6%. Immobilization enhanced the ethanol tolerance of yeast cells. In co-immobilization of S. cerevisiae with A. oryzae or M. purpureus, the optimal hardening time of gel beads was between 15 and 60 min. Bioethanol production was 3.05-3.17% (v v⁻¹) and the Y_E/s (yield of ethanol production/starch consumption) was 0.31-0.37 at pH 4, 30 °C and 150 rpm during 13 days fermentation period. Co-immobilization of S. cerevisiae with a mixed cultures of A. oryzae and M. purpureus at a ratio of 2:1, the bioethanol production was 3.84% (v v⁻¹), and the Y_E/s was 0.39 for a 11 days incubation. However a ratio of A. oryzae and M. purpureus at 1:2 resulted a bioethanol production rate of 4.08% (v v⁻¹), and a Y_E/s of 0.41 after 9 days of fermentation.     

Isolation of a flocculating Saccharomyces cerevisiae and investigation of its performance in the fermentation of beet molasses to ethanol

The present study aims at evaluating the performance of the relative importance of different types of interactions in yeast cell flocculation. The yeasts isolated from several sources, purified and identified by morphological and biochemical methods. About 50 strains of yeast were isolated on PDA medium, among those strains six strains were flocculent and one of them was identified as Saccharomyces cerevisiae. The influence of different additives on the flocculation was investigated and it was shown that pistacia gum (turpentine) increases the flocculation up to 93.8%. Addition of ammonium sulfate (0.2 g l⁻¹) increases the production of alcohol to 6.19%.     

Biodiesel from Milo (Thespesia populnea L.) seed oil

There is a need to seek non-conventional seed oil sources for biodiesel production due to issues such as supply and availability as well as food versus fuel. In this context, Milo (Thespesia populnea L.) seed oil was investigated for the first time as a potential non-conventional feedstock for preparation of biodiesel. This is also the first report of a biodiesel fuel produced from a feedstock containing cyclic fatty acids as T. populnea contains 8,9-methylene-8-heptadecenoic (malvalic) and smaller amounts of two cyclopropane fatty acids besides greater amounts of linoleic, oleic and palmitic acids. The crude oil extracted from T. populnea seed was transesterified under standard conditions with sodium methoxide as catalyst. Biodiesel derived from T. populnea seed oil exhibited fuel properties of density 880 kg m⁻³, kinematic viscosity 4.25 mm²/s; cetane number 59.8; flash point 176 °C; cloud point 9 °C; pour point 8 °C; cold filter plugging point 9 °C; sulfur content 11 mg kg⁻¹; water content 150 mg kg⁻¹; ash content 15 mg kg⁻¹; and acid value as KOH 250 mg kg⁻¹. The oxidative stability of 2.91 h would require the use of antioxidants to meet specifications in standards. Generally, most results compared well with ASTM D6751 and EN 14214 specifications.     

Conversion of paper sludge to ethanol by separate hydrolysis and fermentation (SHF) using Saccharomyces cerevisiae

The conversion of ethanol from paper sludge using the separate hydrolysis and fermentation (SHF) process with cellulase and Saccharomyces cerevisiae GIM-2 were investigated in this paper. Optimization strategy based on statistical experimental designs was employed to enhance degree of saccharification by enzymatic hydrolysis of paper sludge. Based on the Plackett-Burman design, hydrolysis time, substrate concentration and cellulase dosage were selected as the most significant variable on the degree of saccharification. Subsequently, the optimum combination of the selected factors was investigated by a Box-Behnken approach. A mathematical model was developed to show the effects of each factor and their combinatorial interactions on the degree of saccharification. The optimal conditions were hydrolysis time 82.7 h, substrate concentration 40.8 g L⁻¹ and cellulase dosage 18.1 FPU g⁻¹ substrate, and a degree of saccharification of 82.1% can be achieved. When hydrolysate was further fermented with S. cerevisiae GIM-2, the conversion rate of sugar to ethanol was 34.2% and the ethanol yield was 190 g kg⁻¹ of dry paper sludge, corresponding to an overall conversion yield of 56.3% of the available carbohydrates on the initial substrate. The results derived from this study indicate that the response surface methodology is a useful tool for optimizing the hydrolysis conditions to converse paper sludge to ethanol.     

Bioethanol production from sweet sorghum: Evaluation of post-harvest treatments on sugar extraction and fermentation

Three experimental sweet sorghum varieties (M81, Topper and Theis) and three post-harvest conditions were evaluated for ethanol production: juices extracted by milling were obtained from the whole plant, plant without panicle, and stalk (plant without panicle and leaves), respectively. A linear relationship was found between the total fermentable sugar concentrations and Brix degrees of the juices, which can predict the potential ethanol yield by field analytical tests. The juice extractability presented different behavior among the sweet sorghum varieties with respect to the treatments studied. However such treatments did not affect the level of sugar concentration of the juices obtained and the fermentation efficiency. Topper and Theis showed the best performance in terms of ethanol concentration, fermentation efficiency and ethanol yield. The variety used and its post-harvest treatment should be appropriately selected in order to improve the ethanol production from sweet sorghum.     

Antibiotics production of cellulosic waste with solid state fermentation by Streptomyces

Cellulosic waste, corncob, was used as a substrate in the production of oxytetracycline by Streptomyces rimosus TM-55 in solid state fermentation. Oxytetracycline was detected on the fourth day, and reached its maximum on the eighth day. During cultivation, the moisture content of substrate increased as incubation being, and pH value increased slightly. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64 to 67%, supplemented with 20% (w/w) rice bran or 1.5 to 2.5% (NH₄)₂SO₄ as the sole nitrogen source, 1.0% CaCO₃, 2% MgSO₄ 7H₂O, 0.5% KH₂PO₄, and 0.6 to 0.8% aspartic acid or lysine, with incubation for 8 days at 25 to 30°C. Each gram of substrate produced 10 to 11 mg of oxytetracycline.     

Studying the ability of Fusarium oxysporum and recombinant Saccharomyces cerevisiae to efficiently cooperate in decomposition and ethanolic fermentation of wheat straw

Fusarium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae F12 were used to ferment carbohydrates of wet exploded pre-treated wheat straw (PWS) directly to ethanol. Both microorganisms were first grown aerobically to produce cell mass and thereafter fermented PWS to ethanol under anaerobic conditions. During fermentation, soluble and insoluble carbohydrates were hydrolysed by the lignocellulolytic system of F. oxysporum. Mixed substrate fermentation using PWS and corn cobs (CC) in the ratio 1:2 was used to obtain an enzyme mixture with high cellulolytic and hemicellulolytic activities. Under these conditions, activities as high as 34300, 9100, 326, 24, 169, 27 and 254 U dm⁻³ of xylanase, endoglucanase, ??-glucosidase, arabinofuranosidase, avicelase, feruloyl esterase and acetyl esterase, respectively, were obtained. The replacement of the enzyme production phase of F. oxysporum by the addition of commercially available enzymes Celluclast^® 1.5 L FG and Novozym^® 188 in 3:1 ratio for the treatment of PWS, resulted in a 3-fold increase in the volumetric ethanol productivity without increasing the ethanol production significantly. By direct bioconversion of 110 kg m⁻³ dry matter of PWS, ethanol concentration (4.9 kg m⁻³) and yield (40 g kg⁻¹ of PWS) were similarly obtained by F. oxysporum and the mixed culture, while productivity rates as high as 34 g m⁻³ h⁻¹ and 108 g m⁻³ h⁻¹ were obtained by F. oxysporum and the mixed culture, respectively.     

Fermentative hydrogen production with xylose by Clostridium and Klebsiella species in anaerobic batch reactors

Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H₂ and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H₂/mol xylose), 8% (0.5 mol H₂/mol xylose), 10% (0.6 mol H₂/mol xylose) and 14% (0.8 mol H₂/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.     

Improvement of hydrogen production by over-expression of a hydrogen-promoting protein gene in Enterobacter cloacae

The gene of a hydrogen-promoting protein (which we term HPP) from Enterobacter cloacae IIT-BT 08 was cloned and over-expressed in E. cloacae CICC10017 for the first time in this study, and the overall hydrogen yield was greatly improved using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into a hydrogen producing strain of E. cloacae CICC10017 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the GST-tagged protein. An in vitro assay of cell lysates indicates hydrogenase activity of the recombinant strain is 534.78 ± 18.51 ml/(g-DW·h), nearly 2-fold higher than the wild strain. The hydrogen yield of the recombinant strain is 2.55 ± 0.1 mol/mol-glucose, also 2-fold higher than the wild strain. The recombinant strain produces more acetate and butyrate during hydrogen fermentation, but less ethanol, due to the higher hydrogenase activity with the over-expression of the hydrogen-promoting protein. Together, the results demonstrate that successful expression of a single structural gene improves the overall yield of hydrogen by directing metabolic fluxes away from formation of products that compete for NADH.     

Wastewater from monosodium glutamate industry as a low cost fertilizer source for corn (Zea mays L.)

Nitrogen rich wastewater from monosodium glutamate industry (MSG) and paper-mill wastewater were used in this study as low cost fertilizers for growing corn, a common fuel crop. Detailed characterization of the wastewaters and toxicity testes were conducted to assure the safety of these wastewaters. In a greenhouse pot experiment, effects of these wastewaters on corn growth and biomass yield along with the soil properties were evaluated. MSG-wastewater was applied at three rates i.e., zero, 5 m³ ha⁻¹ and 7.5 m³ ha⁻¹ and paper-mill wastewater was applied at and zero, 3.5 m³ ha⁻¹ and 5 m³ ha⁻¹ in a complete randomized blocks design experiment. Significant increase in the corn biomass yield was observed in all the wastewater treatments compared to the Control. Both these wastewaters did not show any adverse effects on plant. N-use efficiency from the MSG-wastewater was comparable to urea-N application. This study emphasizes on sustainable practices for energy crop production by utilizing wastewaters as fertilizer sources. Hence, we report for the first time that the MSG-wastewater can be used for growing corn as a low cost green practice without adverse affects on the soil properties.     

Accelerated startup of biological hydrogen production process by addition of Ethanoligenens harbinense B49 in a biofilm-based column reactor

Two biofilm-based column reactors with walnut shell (WS) as carrier media were applied for fermentative hydrogen production using glucose as substrate by mixed microbial cultures at the temperature of 35 °C. Pure hydrogen producing bacteria Ethanoligenens harbinense B49 was supplemented into the reacting system periodically or continuously to enhance hydrogen production ability at the startup period. The results showed that the bioreactors supplemented with E. harbinense B49 performed better than the reactor without bacteria addition. Continuous addition mode was recommended, since the hydrogen production performance was better and the operation was easily to be accomplished. The optimal addition amount of pure bacteria was also investigated. The optimal bacterial addition amount was found to be 2.5% which led a better hydrogen production rate. In addition, the bioreactor supplemented with pure bacteria continuously presented a high hydrogen production ability as the specific hydrogen production rate (SHPR) maximized at 1.36 L/g-VSS·d; whereas, the bioreactor without bacteria addition obtained a maximum specific hydrogen production rate of 1.10 L/g-VSS·d. The addition of E. harbinense B49 favored the transformation to ethanol type fermentation in the bioreactor. Thereby, the startup period had been accelerated remarkably.     

Energetic potentialities of in vitro cultures of plant cells: Crude oil produced by calli and cells of Euphoria characias

The content of crude oil produced by Euphorbia characias calli vary significantly with the basal medium used. Contrarily to what occurs with in nature growing plants, either calli or suspended cells from this species show a positive correlation between biomass growth and specific crude oil production. Heterotrophic suspended cells from Euphorbia characias at growth exponential phase revealed crude oil contents of 4–5% of dry weight, similar to those found in nature Spring growing plants of this species.     

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Escherichia coli can produce H₂ from glucose via formate hydrogen lyase (FHL). In order to improve the H₂ production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H₂ production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H₂ production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H₂ yield; 1.80 mol/mol glucose under high H₂ pressure or 2.11 mol/mol glucose under reduced H₂ pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdABdirected most of the carbons from glucose to the glycolytic pathway leading to H₂ production by FHL, not to the pentose phosphate pathway.     

Hydrogen production of Enterobacter aerogenes altered by extracellular and intracellular redox states

Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which the substrates are reduced. To examine the effect of intracellular redox state on hydrogen yield, glucose-limiting chemostat cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3. The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound NAD(P)H-dependent hydrogenase.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.     

Remarkable improvement of NOx–PM trade-off in a diesel engine by means of bioethanol and EGR

In order to realize a premixed compression ignition (PCI) engine, the effects of bioethanol–gas oil blends and exhaust gas recirculation (EGR) on PM–NO_x trade-off have been investigated focusing on ignition delay, premixed combustion, diffusion combustion, smoke, NO_x and thermal efficiency. The present experiment was done by increasing the ethanol blend ratio and ethanol and by increasing the EGR ratio in a single cylinder direct injection diesel engine. It is found that a remarkable improvement in PM–NO_x trade-off can be achieved by promoting the premixing based on the ethanol blend fuel having low evaporation temperature, large latent heat and low cetane number as well, in addition, based on a marked elongation of ignition delay due to the low cetane number fuel and the low oxygen intake charge. As a result, very low levels of NO_x and PM, which satisfies the 2009 emission standards imposed on heavy duty diesel engines in Japan, were achieved without deterioration of brake thermal efficiency in the PCI engine fuelled with the 50% ethanol blend diesel fuel and the high EGR ratio. It is noticed that smoke can be reduced even by increasing the EGR ratio under the highly premixed condition.