Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce extracellular cellulase [see 1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

On-line identification of fermentation processes for ethanol production

Bioprocess and Biosystems Engineering - A strategy for monitoring fermentation processes, specifically, simultaneous saccharification and fermentation (SSF) of corn mash, was developed. The...     

Biotechnological production of ethanol: Biochemistry,processes and technologies

The majority of environmental problems arise from the use of conventional energy sources. The liability of such problems along with the reduction of fossil energy resources has led to the global need for alternative renewable energy sources. Using renewable biofuels as energy sources is of remarkable and continuously growing importance. Producing bioethanol through conversion of waste and residual biomass can be a viable and important perspective. In the first part of this review, general concepts, approaches and considerations concerning the utilization of the most important liquid biofuels, namely biodiesel and bioethanol, are presented. Unlike biodiesel (specifically first generation biodiesel), the production of bioethanol is exclusively based on the utilization of microbial technology and fermentation engineering. In the second part of this review, the biochemistry of ethanol production, with regards to the use of hexoses, pentoses or glycerol as carbon sources, is presented and critically discussed. Differences in the glycolytic pathways between the major ethanol‐producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) are presented. Regulation between respiration and fermentation in ethanol‐producing yeasts, viz. effects “Pasteur”, “Crabtree”, “Kluyver” and “Custers”, is discussed. Xylose and glycerol catabolism related with bioethanol production is also depicted and commented. The technology of the fermentation is presented along with a detailed illustration of the substrates used in the process and in pretreatment of lignocellulosic biomass, and the various fermentation configurations employed (separate hydrolysis and fermentation, simultaneous saccharification and fermentation, simultaneous saccharification and co‐fermentation and consolidated bioprocessing). Finally, the production of bioethanol under non‐aseptic conditions is presented and discussed.     

Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes

This study was aimed to study the effect of commercial cellulases (Celluclast 1.5 LFG) on Kluyveromyces marxianus CECT 10875 growth and ethanol production in SSF processes. Preliminary tests carried out in glucose (50 g/L) fermentation medium showed that high enzyme amounts (2.5-3.5 FPU/mL) could cause a negative effect on K. marxianus growth rate and viable cells number. However, the maximum ethanol production was not affected and about 86% of the theoretical (22 g/L) was reached in all cases independently of the enzyme dosage. In SSF experiments, cell viability was always affected by enzyme loading. Nevertheless, slight differences observed on cell viability during glucose fermentation processes with the detected concentrations of the additives did not justify the negative effect observed in SSF experiments.     

Cellulase, clostridia, and ethanol.

Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.     

Comparison of SHF and SSF processes from steam-exploded wheat straw for ethanol production by xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiae strains

In this study, bioethanol production from steam-exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose-fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced.     

Utilization of alkali-treated and neutralized wheat straw-based rations for growing goat kids

Six groups of six goat kids were fed individually for 168 days with wheat straw given various treatments: (1) control; (2) 33 g NaOH/kg straw; (3) 80 g NaOH/kg, partly neutralized with mineral acids; (4) mineral control for 80 g NaOH/kg; (5) 120 g NaOH/kg, partly neutralized with mineral acids, and (6) mineral control for 120 g NaOH/kg straw. The average weight gain was significantly superior (P< 0.05) and the efficiency of dry matter (DM) and energy utilization was the highest with the 80 g NaOH/kg straw treatment. This treatment also gave significantly higher (P<0.05) digestibility of DM, organic matter (OM), neutral detergent fibre (NDF), acid detergent fibre (ADF), nitrogen-free extract (NFE) and hemicellulose than the control and 33 g NaOH/kg straw treatments. Increasing levels of alkali decreased (P<0.05) the digestibility of crude protein (CP) and ether extract (EE). Digestible energy and nitrogen-corrected metabolisable energy (ME_n) (as a percentage of gross energy (GE)) were maximal with 80 g NaOH/kg. The pH value of rumen liquor was the same for the control and the 33 g NaOH/kg and 80 g NaOH/kg treatments, but significantly increased (P<0.05) with the 120 g NaOH/kg straw treatment. The mean values for rumen ammonia nitrogen (NH₃ -N) were the same for the control, the 33 g NaOH/kg, and mineral controls for 80 and 120 g NaOH/kg treatments, but 80 g NaOH and 120 g NaOH/kg straw gave significantly lower values. It is suggested that by partially neutralizing the residual alkali, 80 g NaOH/kg straw can give higher efficiency of energy utilization for growth and digestibility of nutrients compared with 33 g NaOH/kg or the untreated control group, and the extensive use of treated straw in the diets of animals of which a rapid rate of production is not demanded, may be advantageous.     

Kinetics of growth and ethanol production on different carbon substrates using genetically engineered xylose-fermenting yeast

Saccharomyces cerevisiae 424A (LNH-ST) strain was used for fermentation of glucose and xylose. Growth kinetics and ethanol productivity were calculated for batch fermentation on media containing different combinations of glucose and xylose to give a final sugar concentration of 20+/-0.8 g/L. Growth rates obtained in pure xylose-based medium were less than those for media containing pure glucose and glucose-xylose mixtures. A maximum specific growth rate micro(max) of 0.291 h(-1) was obtained in YPD medium containing 20 g/L glucose as compared to 0.206 h(-1) in YPX medium containing 20 g/L xylose. In media containing combinations of glucose and xylose, glucose was exhausted first followed by xylose. Ethanol production on pure xylose entered log phase during the 12-24h period as compared to the 4-10h for pure glucose based medium using 2% inoculum. When glucose was added to fermentation flasks which had been initiated on a pure xylose-based medium, the rate of xylose usage was reduced indicating cosubstrate inhibition of xylose consumption by glucose.     

Integrated production of xylitol and ethanol using corncob

Xylitol production from corncob hemicellulose is a popular process in China. Microbial conversion of xylose to xylitol, as a biological process with many advantages, has drawn increasing attention. As a by-product from the manufacturing of xylitol, corncob cellulosic residues are produced in very large amounts and represent an environmental problem. As a result, considering the large amount of xylitol production in China, the conversion of corncob cellulosic residues has become a widespread issue having to be tackled. After the hemicellulose in corncob has been hydrolyzed for xylitol production, the corncob cellulosic residue is porous and can easily be hydrolyzed by cellulases into glucose and further converted to ethanol, another high-added-value chemical. Based on the latest technology advancements in xylitol, cellulase, and ethanol production, the integrated production of ethanol from corncob cellulosic residues appears as a promising way to improve the profit of the whole xylitol production process.     

Life cycle assessment of energy and GHG emissions during ethanol production from grass straws using various pretreatment processes

Purpose  

The aim of this study was to perform a well-to-pump life cycle assessment (LCA) to investigate the overall net energy balance and environmental impact of bioethanol production using Tall Fescue grass straw as feedstock. The energy requirements and greenhouse gas (GHG) emissions were compared to those of gasoline to explore the potential of bioethanol as sustainable fuel.     

Adsorption characterisation of water and ethanol on wheat starch and wheat gluten using inverse gas chromatography

Adsorption of water and ethanol on wheat starch and wheat gluten has been studied in the temperature range of 60–150 °C using inverse gas chromatography (IGC). From the chromatographic retention data it is able to calculate the separation factors for the two solutes and obtain values for thermodynamic parameters such as Gibbs free energy (ΔG_s) and the enthalpy (ΔH_s) of adsorption of water and ethanol. The results indicate that water is adsorbed more strongly than ethanol at all temperatures, and the low temperature is found to facilitate the adsorptive separation of water from ethanol. It is also shown that the starch definitely plays a crucial role for the water and ethanol separation, despite that wheat flour includes both gluten and starch. The wheat starch is seen to have potential application in biomass water–ethanol separation to obtain fuel ethanol through the preferential adsorption of water from aqueous ethanol.     

Continuous ethanol production using induced yeast aggregates

Summary The induction of yeast cell aggregates in a column reactor was initiated by packing yeast cell paste of Saccharomyces uvarum into the column, and then YMP broth was fed into the column from the bottom at a linear flow rate of 2.5 cm/h. Thereafter, yeast cells aggregated in the column within 48 h without a supply of oxygen. When this yeast aggregate column reactor was used for continuous ethanol production, a final ethanol concentration of 10.8% (w/v) was obtained from 23% (w/v) of glucose in a YMP broth with a dilution rate of 0.05 h^-1, and 4.9% (w/v) was obtained from 10% (w/v) of glucose with a dilution rate of 0.6 h^-1. The theoretical yield was above 97% in both cases. The ethanol production rates were 13 g¹ h^-1 l^-1 and 90 g¹ h^-1 l^-1 for producing 10.8% (w/v) and 4.9% (w/v) of ethanol respectively. This column reactor was maintained at a steady state for more than one month.     

Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products

Pulps with residual lignin ranging from 6.4-27.4% (w/w) were prepared from mixed softwoods using a proprietary biorefining technology (the Lignol process) based on aqueous ethanol organosolv extraction. The pulps were evaluated for bioconversion using enzymatic hydrolysis of the cellulose fraction to glucose and subsequent fermentation to ethanol. All pulps were readily hydrolyzed without further delignification. More than 90% of the cellulose in low lignin pulps (< or =18.4% residual lignin) was hydrolyzed to glucose in 48 h using an enzyme loading of 20 filter paper units/g cellulose. Cellulose in a high lignin pulp (27.4% residual lignin) was hydrolyzed to >90% conversion within 48 h using 40 filter paper units/g. The pulps performed well in both sequential and simultaneous saccharification and fermentation trials indicating an absence of metabolic inhibitors. Chemical and physical analyses showed that lignin extracted during organosolv pulping of softwood is a suitable feedstock for production of lignin-based adhesives and other products due to its high purity, low molecular weight, and abundance of reactive groups. Additional co-products may be derived from the hemicellulose sugars and furfural recovered from the water-soluble stream.     

Bioethanol production from non-starch carbohydrate residues in process streams from a dry-mill ethanol plant

Slurries obtained from process streams in a starch-to-ethanol plant, Agroetanol AB in Norrk?ping, Sweden, were used to assess the potential increase in bioethanol yield if heat treatment followed by enzymatic hydrolysis were applied to the residual starch-free cellulose and hemicellulose fractions. The effects of different pretreatment conditions on flour (the raw material), the stream after saccharification of starch, before fermentation, and after fermentation were studied. The conditions resulting in the highest concentration of glucose and xylose in all streams were heat treatment at 130 degrees C for 40 min with 1% H(2)SO(4). Mass-balance calculations over the fermentation showed that approximately 64%, 54%, 75% and 67% of the glucan, xylan, galactan and arabinan, respectively, in the flour remained water insoluble in the process stream after fermentation without any additional treatment. Utilizing only the starch in the flour would theoretically yield 425 L ethanol per ton flour. By applying heat pretreatment to the water-insoluble material prior to enzymatic hydrolysis, the ethanol yield could be increased by 59 L per ton flour, i.e. a 14% increase compared with starch-only utilization, assuming fermentation of the additional pentose and hexose sugars liberated.     

Leaf litter processing and exoenzyme production on leaves in streams of different pH

We examined microbial colonization, exoenzyme activity, and processing of leaves of yellow poplar (Liriodendron tulipifera), red maple (Acer rubrum), and white oak (Quercus alba) in three streams on the Allegheny Plateau of West Virginia, United States. Leaf packs were placed in streams that varied in their underlying bedrock geology, and therefore in their sensitivity to the high level of acidic precipitation that occurs in this region. The mean pH of the streams was 4.3 in the South Fork of Red Run (SFR), 6.2 in Wilson Hollow Run (WHR), and 7.7 in the North Fork of Hickman Slide Run (HSR). Through time, the patterns of microbial biomass and exoenzyme activity were generally similar among leaf species, but the magnitude of microbial biomass and exoenzyme activity differed among leaf species. Pectinase activity was greatest in HSR, the most alkaline stream, whereas the activity of exocellulase and xylanase was greatest in WHR and SFR, the intermediate and acidic streams. This variation in the activity of different exoenzymes was consistent with published pH optima for these exoenzymes. Variation in processing rates, both among leaf species and among streams, seems to be related to the level of microbial exoenzyme activity on the leaf detritus.     

Durum wheat haploid production using maize wide-crossing

While anther culture or pollinations with Hordeum bulbosum have provided suitable methods for haploid production in bread wheat, they have been largely unsuccessful in durum wheat. Pollinations with maize were used in an attempt to produce haploid seedlings and, from these, fertile doubled haploids of durum wheats. Moreover, the effect of various concentrations and combinations of a synthetic auxin, 2, 4-dichlorophenoxyacetic acid (2,4-D), kinetin, and an ethylene inhibitor, silver nitrate (AgNO₃), on embryo recovery were also investigated. Haploid seedlings were recovered from Triticum turgidum ssp. turgidum cv Rampton Rivet pollinated with maize following in-vivo treatment of ovaries with 2,4-D for 2 weeks and subsequent embryo culture. The recovery of haploid seedlings from T. turgidum ssp. durum cv. Wakona pollinated with maize necessitated the addition of AgNO₃, to the 2,4-D treatment. Overall, haploid seedlings were produced in 1.7% and 3.3% of pollinated florets for Rampton Rivet and Wakona respectively. The success of the present work represents a significant breakthrough for haploid production in durum wheats. Wide hybridization with maize followed by in-vivo treatment of ovaries with 2,4-D alone, or in combination with AgNO₃, may provide a widely-applicable method of haploid production in tetraploid wheats.     

Fractionation of wheat and barley straw to access high-molecular-mass hemicelluloses prior to ethanol production

The cost efficiency of the biorefining process can be improved by extracting high-molecular-mass hemicelluloses from lignocellulosic biomass prior to ethanol production. These hemicelluloses can be used in several high-value-added applications and are likely to be important raw materials in the future. In this study, steam pretreatment in an alkaline environment was used to pretreat the lignocellulosic biomass for ethanol production and, at the same time, extract arabinoxylan with a high-molecular-mass. It was shown that 30% of the arabinoxylan in barley straw could be extracted with high-molecular-mass, without dissolving the cellulose. The cellulose in the solid fraction could then be hydrolysed with cellulase enzymes giving a cellulose conversion of about 80–90% after 72 h. For wheat straw, more than 40% of the arabinoxylan could be extracted with high-molecular-mass and the cellulose conversion of the solid residue after 72 h was about 70–85%. The high cellulose conversion of the pretreated wheat and barley straw shows that they can be used for ethanol production without further treatment. It is therefore concluded that it is possible to extract high-molecular-mass arabinoxylan simultaneously with the pretreatment of biomass for ethanol production in a single steam pretreatment step.     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.     

Trends in biotechnological production of fuel ethanol from different feedstocks

Present work deals with the biotechnological production of fuel ethanol from different raw materials. The different technologies for producing fuel ethanol from sucrose-containing feedstocks (mainly sugar cane), starchy materials and lignocellulosic biomass are described along with the major research trends for improving them. The complexity of the biomass processing is recognized through the analysis of the different stages involved in the conversion of lignocellulosic complex into fermentable sugars. The features of fermentation processes for the three groups of studied feedstocks are discussed. Comparative indexes for the three major types of feedstocks for fuel ethanol production are presented. Finally, some concluding considerations on current research and future tendencies in the production of fuel ethanol regarding the pretreatment and biological conversion of the feedstocks are presented.     

Continuous production of ethanol using immobilized growing yeast cells

Summary Immobilized growing yeast cells were prepared in kappa-carra-geenan gel. Gel beads containing a small number of cells were incubated in a complete medium. The cells grew very well in the gel and the number of living cells per ml of gel increased to over 10 times that of free cells per ml of culture medium. After growing in the gel, the cells formed a dense layer of cells near the gel surface and produced large amounts of ethanol. The conditions for continuous production of ethanol using immobilized growing yeast cells were investigated. The supply of appropriate nutrients for growth was essential for the continuous production. The living cells in the gel were maintained at the high level of 10⁹ per ml of gel and continuous production of ethanol using the complete medium containing 10% glucose was carried out with a retention time of 1 h. In this operation, a stable steady state was maintained for longer than 3 months. The ethanol concentration was 50 mg/ml and the conversion of glucose utilized to ethanol produced was almost 100% of the theoretical yield.