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 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.     

Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae cells immobilized in agar agar and Ca-alginate matrices

Batch fermentation of mahula (Madhuca latifolia L., a tree commonly found in tropical rain forest) flowers was carried out using immobilized cells (in agar agar and calcium alginate) and free cells of Saccharomyces cerevisiae. The ethanol yields were 151.2, 154.5 and 149.1 g kg⁻¹ flowers using immobilized (in agar agar and calcium alginate) and free cells, respectively. Cell entrapment in calcium alginate was found to be marginally superior to those in agar agar (2.2% more) as well as over free cell (3.5% more) as regard to ethanol yield from mahula flowers is concerned. Further, the immobilized cells were physiologically active at least for three cycles [150.6, 148.5 and 146.5 g kg⁻¹ (agar agar) and 152.8, 151.5 and 149.5 g kg⁻¹ flowers (calcium alginate) for first, second and third cycle, respectively] of ethanol fermentation without apparently lowering the productivity. Mahula flowers, a renewable, non-food-grade cheap carbohydrate substrate from non-agricultural environment such as forest can serve as an alternative to food grade sugar/starchy crops such as maize, sugarcane for bio-ethanol production.     

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.     

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.     

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.     

Ethanol production from food residues

Food residues were converted to ethanol by simultaneous saccharification with an amylolytic enzyme complex (a mixture of amyloglucosidase, ??-amylase, and protease), and fermentation (SSF) with the yeast, Saccharomyces cerevisiae. About 36 g dm⁻³ of ethanol was obtained from 100 g dm⁻³ food residue in 48 h of fermentation. In the SSF with no nitrogen supplements, 25 g dm⁻³ of ethanol was produced from 100 g dm⁻³ food residues. In addition, none of the nutrient components except yeast extract from the SSF medium were found to affect ethanol production from food residues. This result indicates that food residues could be a good economic bioresource for ethanol production.     

The effect of nutrient limitation on hydrogen production by batch cultures of Escherichia coli

In order to understand some limiting factors in microbial hydrogen fermentation we have examined hydrogen production by different strains of Escherichia coli grown in batch cultures under different limiting nutrient regimes. The effect of mutations in uptake hydrogenases, in lactate dehydrogenase (ldhA), and fhlA, coding for the regulator of formate hydrogen lyase (fhl) component synthesis, were studied. Each mutation contributed to a modest increase in hydrogen evolution and the effects were synergistic. Various elements were used as limiting nutrient. In batch experiments, limitation for sulfate was without great effect. There was some affect of limiting phosphate with yields approaching 1 mol per mol of glucose. However, strains showed the highest yield of hydrogen per glucose (∼2$\sim 2$) when cultured at limiting concentrations of either ammonia or glucose.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Vegetable oil production potential from Jatropha curcas, Croton megalocarpus, Aleurites moluccana, Moringa oleifera and Pachira glabra: Assessment of renewable energy resources for bio-energy production in Africa

Research on vegetable oil for biofuels in Africa and Asia has focused mainly on Jatropha curcas while other potential oil bearing plants have received little attention. Vegetable oil production potential for five oil bearing plant species namely: Aleurites moluccana, Croton megalocarpus, Jatropha curcas, Moringa oleifera and Pachira glabra were investigated. Nuts and seeds of the plants were collected from the wild and their potential for vegetable oil production assessed in terms of seed/nut acreage yield, seed/nut oil content, harvesting requirement, and upstream processing before vegetable oil recovery. All five varieties were found to contain acceptable but different oil content ranging from 20 to 33% w/w, and seed/nut acreage yield of 3 t ha⁻¹ y⁻¹ to 12.5 t ha⁻¹ y⁻¹. Upstream processing was needed for A. moluccana to break open nuts to release the kernel, and dehulling for both C. megalocarpus and J. curcas to release the seeds, before extracting the vegetable oil, while the seeds of both M. oleifera and P. glabra did not need upstream processing. The Multi-criteria Decision Analysis ranked C. megalocarpus as the plant with the highest vegetable oil production potential of 1.8 t ha⁻¹ y⁻¹ followed by M. oleifera, J. curcas (1 t ha⁻¹ y⁻¹), A. moluccana, and P. glabra. The analysis underlines the need for more studies on C. megalocarpus and M. oleifera for biofuel production in Africa and other regions.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

Genetic variability and divergence studies in seed and oil parameters of mahua (Madhuca longifolia Koenig) J.F. Macribide accessions

Variability in oil content and seed weight of 37 accessions of mahua (Madhuca longifolia Koenig) J.F. Macribide collected from different part of Tamil Nadu, India were assessed. There were significant differences in 100-seed weight and oil parameters, namely, kernel oil %, palmitic acid, stearic acid, oleic acid and linoleic acid. Maximum seed weight (340 g) was recorded in IC554535 and the least weight (100 g) was recorded in IC554545. Kernel oil ranged from minimum of 44.4% in IC554535 to maximum 61.5% in IC556617. The saturated fatty acid i.e palmitic acid and stearic acid ranged from 11.7-25.9% to 19.1-32.2%, respectively. The oleic acid ranged from 32.9 to 48.7% of the total fatty acid while linoleic acid ranged from 9.4 to 15.4%. High heritability was recorded for all the traits studied. It was maximum for palmitic acid (98.6%) and minimum for linoleic acid (95.8%). The Euclidean pairwise dissimilarities were calculated and clustered by UPGMA based SAHN clustering method. Maximum and minimum Euclidean pairwise dissimilarities observed were 7.01 and 0.46, respectively. All the 37 accessions were grouped into three major clusters. The accessions in cluster II had low palmitic acid and high oleic acid while cluster III had high palmitic acid and low oleic acid content. Normalized Mantel statistics (r = 0.8) and principal component analysis supported cluster analysis. Thus on the basis of present findings it is suggested that hybridization between accessions of cluster II and III will result in wide spectrum of variability in subsequent generations for medicinal, edible applications and biodiesel production.     

Ethanol production from enzymatic hydrolysis of sugarcane bagasse pretreated with lime and alkaline hydrogen peroxide

In this work we evaluated ethanol production from enzymatic hydrolysis of sugarcane bagasse. Two pretreatments agents, lime and alkaline hydrogen peroxide, were compared in their performance to improve the susceptibility of bagasse to enzymatic action. Mild conditions of temperature, pressure and absence of acids were chosen to diminish costs and to avoid sugars degradation and consequent inhibitors formation. The bagasse was used as it comes from the sugar/ethanol industries, without grinding or sieving, and hydrolysis was performed with low enzymes loading (3.50 FPU g⁻¹ dry pretreated biomass of cellulase and 1.00 CBU g⁻¹ dry pretreated biomass of ??-glucosidase). The pretreatment with alkaline hydrogen peroxide led to the higher glucose yield: 691 mg g⁻¹ of glucose for pretreated bagasse after hydrolysis of bagasse pretreated for 1 h at 25 °C with 7.35% (v/v) of peroxide. Fermentation of the hydrolyzates from the two pretreatments were carried out and compared with fermentation of a glucose solution. Ethanol yields from the hydrolyzates were similar to that obtained by fermentation of the glucose solution. Although the preliminary results obtained in this work are promising for both pretreatments considered, reflecting their potential for application, further studies, considering higher biomass concentrations and economic aspects should be performed before extending the conclusions to an industrial process.     

Remarkable enhancement on hydrogen production performance of Rhodobacter sphaeroides by disrupting spbA and hupSL genes

The redox balance and bacteriochlorophyll (Bchl) synthesis are both significant to hydrogen generation in photosynthetic bacteria. In this study, spbA and hupSL genes were knocked out from the genome of Rhodobacter sphaeroides HY01. The UV–vis spectra showed that the Bchl contents of spbA mutants were enhanced under photosynthetic conditions. The hydrogen yields of WH04 (hupSL⁻) and WSH10 (spbA⁻, hupSL⁻) mutants increased by 19.4%, 21.8%, and the maximum hydrogen evolution rates increased by 29.9% and 55.0% respectively using glutamate as sole nitrogen source. The maximum hydrogen production rate of WSH10 was up to 141.9 mL/(L·h). The nifH expression levels of the mutants and the wild type supported the correlation between hydrogen production and nitrogenase activity. The results demonstrate that disruption of spbA in R. sphaeroides can partially derepress the ammonium inhibition in nitrogenase activity, and indicate that spbA is a negative regulator in nitrogenase synthesis in the presence of ammonium.     

Optimal fermentation conditions for maximizing the ethanol production by Kluyveromyces fragilis from cheese whey powder

Cheese whey powder (CWP) is an attractive raw material for ethanol production since it is a dried and concentrated form of CW and contains lactose in addition to nitrogen, phosphate and other essential nutrients. In the present work, deproteinized CWP was utilized as fermentation medium for ethanol production by Kluyveromyces fragilis. The individual and combined effects of initial lactose concentration (50-150 kg m⁻³), temperature (25-35 °C) and inoculum concentration (1-3 kg m⁻³) were investigated through a 2³ full-factorial central composite design, and the optimal conditions for maximizing the ethanol production were determined. According to the statistical analysis, in the studied range of values, only the initial lactose concentration had a significant effect on ethanol production, resulting in higher product formation as the initial substrate concentration was increased. Assays with initial lactose concentration varying from 150 to 250 kg m⁻³ were thus performed and revealed that the use of 200 kg m⁻³ initial lactose concentration, inoculum concentration of 1 kg m⁻³ and temperature of 35 °C were the best conditions for maximizing the ethanol production from CWP solution. Under these conditions, 80.95 kg m⁻³ of ethanol was obtained after 44 h of fermentation.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation

The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.