Analysis of sugars by liquid chromatography-mass spectrometry in Jerusalem artichoke tubers for bioethanol production optimization

Sustainability of biofuels is increasingly taken into account; therefore, sustainable production technologies are needed. There has been a long history of converting Jerusalem artichoke into ethanol. Jerusalem artichoke (Helianthus tuberosus L.) is a low-requirement crop, it has a high carbohydrates yield and, nowadays, it does not interfere with food chain. It is, then, a promising energy crop for sustainable bioethanol production. However, the main storage carbohydrate of Jerusalem artichoke, inulin, can not be directly fermented by classic fermentation yeasts, so, either a hydrolysis followed by fermentation with classical yeast or the use of yeasts with inulinase activity are required to obtain bioethanol. Therefore, it is needed to know not only total sugar content, but also their composition, for the bioethanol production optimization from Jerusalem artichoke tubers. Several methods have been used in literature for carbohydrates analysis present in Jerusalem artichoke tubers. However, for further development of carbohydrate analysis, faster and more reliable identification and peak confirmation, mass spectrometry (MS) detection is required. In this paper, liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) was used as an alternative technique to analyse sugars content and composition in tubers from Jerusalem artichoke. Two simple, rapid, sensitive and specific LC-ESI-MS methods were developed under the positive ionization mode. Glucose, fructose, sucrose, kestose and inulin were determined. Furthermore, inulin profile can be characterized. Analytical reversed phase LC columns were used using only water as eluent. These methods can be useful to optimize the whole bioethanol production chain from Jerusalem artichoke.     

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.     

Optimization of bioethanol production from glycerol by Escherichia coli SS1

Bioethanol is a promising biofuel and has a lot of great prospective and could become an alternative to fossil fuels. Ethanol fermentation using glycerol as carbon source was carried out by local isolate, ethanologenic bacterium Escherichia coli SS1 in a close system. Factors affecting bioethanol production from pure glycerol were optimized via response surface methodology (RSM) with central composite design (CCD). Four significant variables were found to influence bioethanol yield; initial pH of fermentation medium, substrate concentration, salt content and organic nitrogen concentration with statistically significant effect (p ≤ 0.05) on bioethanol production. The significant factor was then analyzed using central composite design (CCD). The optimum conditions for bioethanol production were substrate concentration at 34.5 g/L, pH 7.61, and organic nitrogen concentration at 6.42 g/L in which giving ethanol yield approximately 1.00 mol/mol. In addition, batch ethanol fermentation in a 2 L bioreactor was performed at the glycerol concentration of 20 g/L, 35 g/L and 45 g/L, respectively. The ethanol yields obtained from all tested glycerol concentrations were approaching theoretical yield when the batch fermentation was performed at optimized conditions.     

Optimization of very high gravity fermentation process for ethanol production from industrial sugar beet syrup

In order to reduce production costs and environmental impact of bioethanol from sugar beet low purity syrup 2, an intensification of the industrial alcoholic fermentation carried out by Saccharomyces cerevisiae is necessary. Two fermentation processes were tested: multi-stage batch and fed-batch fermentations with different operating conditions. It was established that the fed-batch process was the most efficient to reach the highest ethanol concentration. This process allowed to minimize both growth and ethanol production inhibitions by high sugar concentrations or ethanol. Thus, a good management of the operating conditions (initial volume and feeding rate) could produce 15.2% (v/v) ethanol in 53 h without residual sucrose and with an ethanol productivity of 2.3 g L h⁻¹.     

Comparison of entrapment and biofilm mode of immobilisation for bioethanol production from oilseed rape straw using Saccharomyces cerevisiae cells

Cell immobilisation provides the opportunity to reduce the cost of producing bioethanol from lignocellulosic biomass such as oilseed rape (OSR) straw, in addition to enhancing operational stability. Bioethanol fermentation of OSR straw hydrolysate by free and immobilised Saccharomyces cerevisiae was studied. Cells were either entrapped in alginate beads or Lentikat^® discs or immobilised as a biofilm on spent grains, Leca, or reticulated foam. The overall aims of the research were to compare bioethanol yields produced from free and immobilised systems, and to identify the most suitable immobilisation technique in terms of bioethanol yield and longevity of the immobilised cell system. Cell entrapment in alginate beads and Lentikat^® discs resulted in significantly higher bioethanol yields compared to when cells were free in suspension or immobilised as a biofilm on a support material. The maximum amount of bioethanol produced by cells immobilised in alginate beads and Lentikat^® discs were 169.26 ± 0.24 and 165.13 ± 0.67 g bioethanol kg⁻¹ OSR straw after 3 h and 7 h of fermentation, respectively. Due to the high mechanical stability and bioethanol yield, immobilisation of S. cerevisiae in Lentikat^® discs was considered the most appropriate immobilisation technique for bioethanol production.     

Simultaneous utilization of galactose and glucose by Saccharomyces cerevisiae mutant strain for ethanol production

Red algal biomass is a promising alternative feedstock for bioethanol production, due to several advantages including high carbohydrate content, growth rate, ethanol yield, and CO₂ fixation ability. However, it has been known that most yeast strains can not utilize galactose, the major sugar of red algae, as efficiently it can utilize glucose. The authors report a novel ethanogenic strain capable of fermenting galactose, Saccharomyces cerevisiae. This mutant yeast strain exhibited exceptional fermentative performance on galactose and a mixture of galactose and glucose. At 120 g/L of initial galactose concentration, ethanol concentration reached 6.9% (v/v) within 36 h with 88.3% of theoretical ethanol yield (0.51 g ethanol/g galactose). The ethanol concentration and yield were higher than that for glucose at the same initial concentration. In a mixed sugar (galactose + glucose) condition, the existence of glucose retarded galactose utilization however, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose concentration. The critical inhibitory levels of formic acid, levulinic acid and 5-hydroxymethylfurfural (5-HMF) on ethanol fermentation were 0.5, 2.0, and 10.0 g/L; respectively. From this result, the ethanol fermentation efficiency of the novel S. cerevisiae strain using the galactose base of red algae was superior to the fermentation efficiency when using the wild type strain, and the novel strain was found to have resistance to the major inhibitors generated during the saccharification process.     

Efficient ethanol production from inulin by two-stage aerate strategy

A major concern for ethanol production from inulin-containing materials, is the higher unconverted sugar, which increases the cost of ethanol production and wastewater treatment. Some key factors, such as inulinase, biomass or aeration rates, were studied to solve the problems in the process of ethanol fermentation from inulin. It was showed that more inulinase and increasing inoculum size can shorten the fermentation time, but could not reduce residual sugars. Two-stage aerate strategy was developed to utilize the remained sugars: keep the aeration at 5 h⁻¹ at the first 12 h, and drop it to 1.2 h⁻¹. Under this condition, contradiction between fermentation time and high ethanol yield was solved (60 h and 0.43 g g⁻¹), and the final residual sugar concentration decreased to about 10 g L⁻¹ with 98 g L⁻¹ ethanol. The ethanol productivity was up to 1.63 g L⁻¹ h⁻¹, which is the highest productivity of ethanol fermentations from inulin-containing materials.     

菊芋生产燃料乙醇工艺路线探讨

在对菊芋汁清液连续发酵和菊芋粉带渣批式发酵的研究基础上,进行了利用菊芋生产燃料乙醇的两种工艺路线的技术经济可行性分析,提出建立分布式加工体系的策略。菊芋乙醇产业生态系统包括种植、仓储、生产粗乙醇和生产精乙醇4个主生产环节以及副产物综合利用环节。菊芋综合利用路线的提出,期望能够为降低生产成本,实现菊芋原料生产燃料乙醇的产业化提供依据。     

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.     

Arrowroot as a novel substrate for ethanol production by solid state simultaneous saccharification and fermentation

Ethanol production from Canna edulis Ker was successfully carried out by solid state simultaneous saccharification and fermentation. The enzymatic hydrolysis conditions of C. edulis were optimized by Plackett–Burman design. The effect of inert carrier (corncob and rice bran) on ethanol fermentation and the kinetics of solid state simultaneous saccharification and fermentation was investigated. It was found that C. edulis was an alternative substrate for ethanol production, 10.1% (v/v) of ethanol concentration can attained when 40 g corncob and 10 g rice bran per 100 g C. edulis powder were added for ethanol fermentation. No shortage of fermentable sugars was observed during solid state simultaneous saccharification and fermentation. There was no wastewater produced in the process of ethanol production from C. edulis with solid state simultaneous saccharification and fermentation and the ethanol yield of more than 0.28 tonne per one tonne feedstock was achieved. This is first report for ethanol production from C. edulis powder.     

Process optimization for bioethanol production from cassava starch using novel eco-friendly enzymes

Although cassava (Manihot esculenta Crantz) is a potential bioethanol crop, high operational costs resulted in a negative energy balance in the earlier processes. The present study aimed at optimizing the bioethanol production from cassava starch using new enzymes like Spezyme^® Xtra and Stargen™ 001. The liquefying enzyme Spezyme was optimally active at 90 °C and pH 5.5 on a 10% (w/v) starch slurry at levels of 20.0 mg (280 Amylase Activity Units) for 30 min. Stargen levels of 100 mg (45.6 Granular Starch Hydrolyzing Units) were sufficient to almost completely hydrolyze 10% (w/v) starch at room temperature (30 ± 1 °C). Ethanol yield and fermentation efficiency were very high (533 g/kg and 94.0% respectively) in the Stargen + yeast process with 10% (w/v) starch for 48 h. Raising Spezyme and Stargen levels to 560 AAU and 91.2 GSHU respectively for a two step loading [initial 20% (w/v) followed by 20% starch after Spezyme thinning]/initial higher loading of starch (40% w/v) resulted in poor fermentation efficiency. Upscaling experiments using 1.0 kg starch showed that Stargen to starch ratio of 1:100 (w/w) could yield around 558 g ethanol/kg starch, with a high fermentation efficiency of 98.4%. The study showed that Spezyme level beyond 20.0 mg for a 10% (w/v) starch slurry was not critical for optimizing bioethanol yield from cassava starch, although an initial thinning of starch for 30 min by Spezyme facilitated rapid saccharification-fermentation by Stargen + yeast system. The specific advantage of the new process was that the reaction could be completed within 48.5 h at 30 ± 1 °C.     

Evaluation of dilute acid and alkaline pretreatments,enzymatic hydrolysis and fermentation of napiergrass for fuel ethanol production

Napiergrass (Pennisetum purpureum Schum.) is a promising low cost raw material which does not compete with food prices, has attractive yields and an environmentally friendly farming. Dilute sulfuric acid pretreatment of napiergrass was effective to obtain high yields of sugars and low level of degradation by-products from hemicellulose. Detoxification with Ca(OH)₂ removed inhibitors but showed sugars loss. An ethanol concentration of 21 g/L after 176 h was found from the hydrolyzate using Pichia stipitis NBRC 10063 (fermentation efficiency 66%). An additional alkaline pretreatment applied to the solid fraction remaining from the diluted acid pretreatment improved the lignin removal. The highest cellulose hydrolysis values were found with the addition of β-glucosidase and PEG 6000. The simultaneous hydrolysis and fermentation of the cellulosic fraction with Saccharomyces cerevisiae, 10% (w/v) solid concentration, β-glucosidase and PEG 6000, showed the highest ethanol concentration (24 g/L), and cellulose hydrolysis values (81%). 162 L ethanol/t of dry napiergrass were produced (overall efficiency of 52%): 128 L/t from the cellulosic fraction and 34 L/t from the hemicellulosic fraction.     

Biological abatement of inhibitors in rice hull hydrolyzate and fermentation to ethanol using conventional and engineered microbes

Microbial inhibitors arise from lignin, hemicellulose, and degraded sugar during pretreatment of lignocellulosic biomass. The fungus Coniochaeta ligniaria NRRL30616 has native ability to metabolize a number of these compounds, including furan and aromatic aldehydes known to act as inhibitors toward relevant fermenting microbes. In this study, C. ligniaria was used to metabolize and remove inhibitory compounds from pretreated rice hulls, which comprise a readily available agricultural residue rich in glucose (0.32–0.33 g glucan/g hulls) and xylose (0.15–0.19 g xylan/g hulls). Samples were dilute-acid pretreated and subjected to bioabatement of inhibitors by C. ligniaria. The bioabated rice hull hemicellulose hydrolyzates were then utilized for ethanol fermentations. In bioabated liquors, glucose was converted to 0.58% (w/v) ethanol by Saccharomyces cerevisiae D5a at 100% of theoretical yield, while fermentations of unabated hydrolyzates either failed to exit lag phase or had reduced ethanol yield (80% of theoretical). In fermentations using ethanologens engineered for conversion of pentoses, bioabatement of hydrolyzates similarly improved fermentations. Fermentation of xylose and arabinose by ethanologenic Escherichia coli FBR5 yielded 2.25% and 0.05% (w/v) ethanol from bioabated and unabated samples, respectively. Fermentations using S. cerevisiae YRH400 had decreased fermentation lag times in bioabated hydrolyzates. However, xylose metabolism in S. cerevisiae YRH400 was strongly affected by pH and acetate concentration.     

Carob pod as a feedstock for the production of bioethanol in Mediterranean areas

There is a growing interest worldwide to find out new and cheap carbohydrate sources for production of bioethanol. In this context, carob pod (Ceratonia siliqua) is proposed as an economical source for bioethanol production, especially, in arid regions. The carob tree is an evergreen shrub native to the Mediterranean region, cultivated for its edible seed pods and it is currently being reemphasised as an alternative in dryland areas, because no carbon-enriched lands are necessary. In this work, the global process of ethanol production from carob pod was studied. In a first stage, aqueous extraction of sugars from the pod was conducted, achieving very high yields (>99%) in a short period of time. The process was followed by acid or alkaline hydrolysis of washed pod at different operating conditions, the best results (R = 38.20%) being reached with sulphuric acid (2% v/v) at 90 °C, using a L/S (liquid/solid) ratio of 7.5 and shaking at 700 rpm for 420 min. After that, fermentation of hydrolysates were tested at 30 °C, 125 rpm, 200 g/L of sugars and 15 g/L of yeast with three different kinds of yeasts. In these conditions a maximum of 95 g/L of ethanol was obtained after 24 h. Finally, the distillation and dehydration of water–bioethanol mixtures was analyzed using the chemical process simulation software CHEMCAD with the aim of estimate the energy requirements of the process.     

Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp. cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase, Avicelase and ??-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase, Avicelase, and ??-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase, exoglucanase and ??-glucosidase resulted in efficient hydrolysis of cellulosic substrate. After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol was obtained within 3 d. The results have significant implications and future applications regarding to production of fuel ethanol from agricultural cellulosic waste.     

Bioethanol production by Saccharomyces cerevisiae,Pichia stipitis and Zymomonas mobilis from delignified coconut fibre mature and lignin extraction according to biorefinery concept

In search to increase the offer of liquid, clean, renewable and sustainable energy in the world energy matrix, the use of lignocellulosic materials (LCMs) for bioethanol production arises as a valuable alternative. The objective of this work was to analyze and compare the performance of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the production of bioethanol from coconut fibre mature (CFM) using different strategies: simultaneous saccharification and fermentation (SSF) and semi-simultaneous saccharification and fermentation (SSSF). The CFM was pretreated by hydrothermal pretreatment catalyzed with sodium hydroxide (HPCSH). The pretreated CFM was characterized by X-ray diffractometry and SEM, and the lignin recovered in the liquid phase by FTIR and TGA. After the HPCSH pretreatment (2.5% (v/v) sodium hydroxide at 180 °C for 30 min), the cellulose content was 56.44%, while the hemicellulose and lignin were reduced 69.04% and 89.13%, respectively. Following pretreatment, the obtained cellulosic fraction was submitted to SSF and SSSF. Pichia stipitis allowed for the highest ethanol yield – 90.18% – in SSSF, 91.17% and 91.03% were obtained with Saccharomyces cerevisiae and Zymomonas mobilis, respectively. It may be concluded that the selection of the most efficient microorganism for the obtention of high bioethanol production yields from cellulose pretreated by HPCSH depends on the operational strategy used and this pretreatment is an interesting alternative for add value of coconut fibre mature compounds (lignin, phenolics) being in accordance with the biorefinery concept.     

Optimization and analysis of a bioethanol agro-industrial system from sweet sorghum

The use of non-food crops for bioethanol production represents an important trend for renewable energy in China. In this paper, a bioethanol agro-industrial system with distributed fermentation plants from sweet sorghum is presented. The system consists of the following processes: sweet sorghum cultivation, crude ethanol production, ethanol refining and by-product utilization. The plant capacities of crude ethanol and pure ethanol, in different fractions of useful land, are optimized. Assuming a minimum cost of investment, transport, operation and so on, the optimum capacity of the pure ethanol factory is 50,000 tonnes/year. Moreover, this bioethanol system, which requires ca. 13,300 ha (hectares) of non-cultivated land to supply the raw materials, can provide 26,000 jobs for rural workers. The income from the sale of the crops is approximately 71 million RMB Yuan and the ethanol production income is approximately 94 million RMB Yuan. The potential savings in CO₂ emissions are ca. 423,000 tonnes/year and clear economic, social and environmental benefits can be realized.     

Photo-fermentative hydrogen production by Rhodopseudomonas palustris CGA009 in the presence of inhibitory compounds

This study investigated Rhodopseudomonas palustris CGA009 biohydrogen production from compounds commonly found in lignocellulosic steam explosion hydrolysate, by examining the effect of individual inhibitory phenolic and furan compounds found in hydrolysates, under photo-fermentative anaerobic conditions. Since lignocellulose is often converted into ethanol via yeast-mediated fermentation, the tolerance of R. palustris CGA009 towards ethanol inhibition was also tested at a concentration range of 0.25–14% (v/v) under anaerobic photo-fermentative conditions. Hydrogen production was enhanced by compounds such as syringaldehyde (0.03 g/L), which accumulated total hydrogen of 960 mL over the cultivation period. In contrast, a reduction in hydrogen production of 1.4 fold was observed in vanillin-containing solutions (0.43 g/L), which obtained accumulated total hydrogen of 576 mL. Increasing ethanol concentrations reduced hydrogen production, but cell growth was not affected up to 1% (v/v), a fairly low concentration. R. palustris CGA009 can tolerate comparatively high concentrations of phenolic compounds, suggesting its use for lignocellulose hydrolysate detoxification and hydrogen production.     

Pretreatment of cassava stems and peelings by thermohydrolysis to enhance hydrolysis yield of cellulose in bioethanol production process

The potential of wastes obtained from the cultivation of Manihot esculenta Crantz as raw material for bioethanol production was studied. The objective was to determine the optimal conditions of hemicellulose thermohydrolysis of cassava stems and peelings and evaluate their impact on the enzymatic hydrolysis yield of cellulose. An experimental design was conducted to model the influence of factors on the pentose, reducing sugar and phenolic compound contents. Residues obtained from the optimal pretreatment conditions were hydrolysed with cellulase (filter paper activity 40 FPU/g). The hydrolysates from pretreatment and enzymatic hydrolysis were fermented respectively using Rhyzopus spp. and Sacharomyces cerevisiae. The yield of enzymatic hydrolysis obtained under the optimal conditions were respectively 73.1% and 86.6% for stems and peelings resulting in an increase of 39.84% and 55.40% respectively as compared to the non-treated substrates. The ethanol concentrations obtained after fermentation of enzymatic hydrolysates were 1.3 and 1.2 g/L respectively for the stem and peeling hydrolysates. The pentose and phenolic compound concentrations obtained from the multi-response optimization were 10.2 g/L; 0.8 g/L and 10.1 g/L; 1.3 g/L respectively for stems and peelings. The hydrolysates of stems and peelings under these optimal conditions respectively gave ethanol concentrations of 5.27 g/100 g for cassava stems and 2.6 g/100 g for cassava peelings.     

Exploring alkaline pre-treatment of microalgal biomass for bioethanol production

We have investigated, for the first time, the alkaline pre-treatment of microalgal biomass, from the species Chlorococcum infusionum, using NaOH for bioethanol production. This pre-treatment step aims to release and breakdown entrapped polysaccharides in the microalgae cell walls into fermentable subunits. Three parameters were examined here; the concentration of NaOH, temperature and the pre-treatment time. The bioethanol concentration, glucose concentration and the cell size were studied in order to determine the effectiveness of the pre-treatment process. Microscopic analysis was performed to confirm cell rupturing, the highest glucose yield was determined to be 350 mg/g, and the maximum bioethanol yield obtained was 0.26 g ethanol/g algae using 0.75% (w/v) of NaOH and 120 °C for 30 min. Overall, the alkaline pre-treatment method proved to be promising option to pre-treat microalgal biomass for bioethanol production.