An integrated platform for gas-diffusion separation and electrochemical determination of ethanol on fermentation broths

An integrated platform was developed for point-of-use determination of ethanol in sugar cane fermentation broths. Such analysis is important because ethanol reduces its fuel production efficiency by altering the alcoholic fermentation step when in excess. The custom-designed platform integrates gas diffusion separation with voltammetric detection in a single analysis module. The detector relied on a Ni(OH)₂-modified electrode. It was stabilized by uniformly depositing cobalt and cadmium hydroxides as shown by XPS measurements. Such tests were in accordance with the hypothesis related to stabilization of the Ni(OH)₂ structure by insertion of Co²⁺ and Cd²⁺ ions in this structure. The separation step, in turn, was based on a hydrophobic PTFE membrane, which separates the sample from receptor solution (electrolyte) where the electrodes were placed. Parameters of limit of detection and analytical sensitivity were estimated to be 0.2% v/v and 2.90 μA % (v/v)⁻¹, respectively. Samples of fermentation broth were analyzed by both standard addition method and direct interpolation in saline medium based-analytical curve. In this case, the saline solution exhibited ionic strength similar to those of the samples intended to surpass the tonometry colligative effect of the samples over analyte concentration data by attributing the reduction in quantity of diffused ethanol vapor majorly to the electrolyte. The approach of analytical curve provided rapid, simple and accurate analysis, thus contributing for deployment of point-of-use technologies. All of the results were accurate with respect to those obtained by FTIR method at 95% confidence level.     

Detoxification and Fermentation of Pyrolytic Sugar for Ethanol Production

The sugars present in bio-oil produced by fast pyrolysis can potentially be fermented by microbial organisms to produce cellulosic ethanol. This study shows the potential for microbial digestion of the aqueous fraction of bio-oil in an enrichment medium to consume glucose and produce ethanol. In addition to glucose, inhibitors such as furans and phenols are present in the bio-oil. A pure glucose enrichment medium of 20?g/l was used as a standard to compare with glucose and aqueous fraction mixtures for digestion. Thirty percent by volume of aqueous fraction in media was the maximum additive amount that could be consumed and converted to ethanol. Inhibitors were removed by extraction, activated carbon, air stripping, and microbial methods. After economic analysis, the cost of ethanol using an inexpensive fermentation medium in a large scale plant is approximately $14 per gallon.     

Discussion of parameters associated with the determination of arsenic by electrothermal atomic absorption spectrometry in slurried environmental samples

A slurry sampling – fast program procedure has been developed for the determination of arsenic in plants, soils and sediments by electrothermal atomic absorption spectrometry. Efficiencies of various single and mixed modifiers for thermal stabilization of arsenic and for a better removal of the matrix during pyrolysis step were compared. The influence of the slurry concentration, amounts of modifier and parameters of the pyrolysis step on the As integrated absorbance signals have been studied and a comparison between fast and conventional furnace programs was also made. The ultrasonic agitation of the slurry followed by a fast electrothermal program using an Ir/Mg modifier provides the most consistent performance in terms of precision and accuracy. The reliability of the whole procedure has been compared with results obtained after application of a wet digestion method with an HF step and validated by analyzing eleven certified reference materials. Arsenic detection and quantitation limits expressed on dry sample matter were about 30 and 100 μg kg^–1, respectively.     

Discussion of parameters associated with the determination of arsenic by electrothermal atomic absorption spectrometry in slurried environmental samples

A slurry sampling-fast program procedure has been developed for the determination of arsenic in plants, soils and sediments by electrothermal atomic absorption spectrometry. Efficiencies of various single and mixed modifiers for thermal stabilization of arsenic and for a better removal of the matrix during pyrolysis step were compared. The influence of the slurry concentration, amounts of modifier and parameters of the pyrolysis step on the As integrated absorbance signals have been studied and a comparison between fast and conventional furnace programs was also made. The ultrasonic agitation of the slurry followed by a fast electrothermal program using an Ir/Mg modifier provides the most consistent performance in terms of precision and accuracy. The reliability of the whole procedure has been compared with results obtained after application of a wet digestion method with an HF step and validated by analyzing eleven certified reference materials. Arsenic detection and quantitation limits expressed on dry sample matter were about 30 and 100 micrograms kg-1, respectively.     

Monitoring of substrate and product concentrations in acetic fermentation processes for onion vinegar production by NIR spectroscopy: value addition to worthless onions

Wastes and by-products of the onion-processing industry pose an increasing disposal and environmental problem and represent a loss of valuable sources of nutrients. The present study focused on the production of vinegar from worthless onions as a potential valorisation route which could provide a viable solution to multiple disposal and environmental problems, simultaneously offering the possibility of converting waste materials into a useful food-grade product and of exploiting the unique properties and health benefits of onions. This study deals specifically with the second and definitive step of the onion vinegar production process: the efficient production of vinegar from onion waste by transforming onion ethanol, previously produced by alcoholic fermentation, into acetic acid via acetic fermentation. Near-infrared spectroscopy (NIRS), coupled with multivariate calibration methods, has been used to monitor the concentrations of both substrates and products in acetic fermentation. Separate partial least squares (PLS) regression models, correlating NIR spectral data of fermentation samples with each kinetic parameter studied, were developed. Wavelength selection was also performed applying the iterative predictor weighting–PLS (IPW-PLS) method in order to only consider significant spectral features in each model development to improve the quality of the final models constructed. Biomass, substrate (ethanol) and product (acetic acid) concentration were predicted in the acetic fermentation of onion alcohol with high accuracy using IPW-PLS models with a root-mean-square error of the residuals in external prediction (RMSEP) lower than 2.5% for both ethanol and acetic acid, and an RMSEP of 6.1% for total biomass concentration (a very satisfactory result considering the relatively low precision and accuracy associated with the reference method used for determining the latter). Thus, the simple and reliable calibration models proposed in this study suggest that they could be implemented in routine applications to monitor and predict the key species involved in the acetic fermentation of onion alcohol, allowing the onion vinegar production process to be controlled in real time.     

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.     

The future of coproducts from corn processing

Increased demand for ethanol as a fuel additive has resulted in dramatic growth in ethanol production. Ethanol is produced from corn by either wet milling or dry-grind processing. In wet milling, the corn kernel is fractionated into different components, resulting in several coproducts. Wet-milling plants are capital intensive because of equipment requirements; they produce large volumes of ethanol and are corporate owned. In dry-grind processing, the corn kernel is not fractionated and only one coproduct, distillers’ dried grains with solubles (DDGS), is generated. Dry-grind plants require less equipment and capital than wet mills. They generate smaller volumes of ethanol, are producer owned, and add direct benefits to rural economies. Most of the increase in ethanol production during the past decade is attributed to growth in the dry-grind industry. The marketing of coproducts provides income to offset processing costs. For dry-grind plants, this is especially important, because only one coproduct is available. Several issues could affect DDGS marketing. The increasing volume of DDGS accompanying ethanol production could reduce market value; high phosphorous content could limit the use of DDGS, because of animal waste disposal issues. Water removal is a costly processing step and affects the economics of ethanol processing. Technologies to remove germ and fiber from DDGS could produce a new coproduct suitable for feeding to nonruminants; this would expand the markets for DDGS. Reducing phosphorus in DDGS would sustain markets for conventional DDGS. The development of more efficient methods of water removal would increase the efficiency of ethanol processing and reduce the costs of processing. New technologies could contribute to greater stability of dry-grind plants.     

甜高粱茎秆生产燃料乙醇

本文对甜高粱茎秆原料的贮藏、汁液液态发酵、茎秆直接粉碎固态发酵以及榨汁后剩余的秆渣预处理同步糖化发酵4个方面的研究情况进行了综述,重点论述了甜高粱茎秆生产燃料乙醇的瓶颈问题即原料的贮藏和秆渣木质纤维素预处理技术。提出了一种更经济合理的甜高粱茎秆生产燃料乙醇工艺流程。     

Corn steep liquor as a cost-effective nutrition adjunct in high-performanceZymomonas ethanol fermentations

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-0-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) byEscherichia coli B (ATCC 11303) and two different ethanologenic recombinants—a strain in whichpet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-typeE. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.     

Optimization of Saccharification and Fermentation Process in Bioethanol Production from Oil Palm Fronds

Oil Palm Frond (OPF) is one of lignocellulosic biomass, which can be utilized as raw material for bioethanol production. Bioethanol is produced as alternative energy to substitute gasoline. There are four steps in bioethanol production from OPF, i.e pretreatement, saccharification, fermentation and purification process. In this study, optimization of saccharification and fermentation process for OPF was investigated. Two methods and the variations of enzyme concentration were carried out in the saccharification and fermentation process. Separate hydrolysis and fermentation process (SHF) and simultaneous saccharification and fermentation process (SSF) were conducted to produce ethanol optimally. Variations of enzyme concentration used in this process were 10, 20, 30 and 40 FPU/g substrate. The result shows that the highest ethanol concentration can be obtained in SSF process with 30 FPU/g substrate of enzyme concentration. The process produced 59.20 g/L ethanol (95.95% yield ethanol) at 96 h of SSF process.     

Advances and Developments in Strategies to Improve Strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and Processes to Obtain the Lignocellulosic Ethanol−A Review

The conversion of biomass into ethanol using fast, cheap, and efficient methodologies to disintegrate and hydrolyse the lignocellulosic biomass is the major challenge of the production of the second-generation ethanol. This revision describes the most relevant advances on the conversion process of lignocellulose materials into ethanol, development of new xylose-fermenting strains of Saccharomyces cerevisiae using classical and modern genetic tools and strategies, elucidation of the expression of some complex industrial phenotypes, tolerance mechanisms of S. cerevisiae to lignocellulosic inhibitors, monitoring and strategies to improve fermentation processes. In the last decade, numerous engineered pentose-fermenting yeasts have been developed using molecular biology tools. The increase in the tolerance of S. cerevisiae to inhibitors is still an important issue to be exploited. As the industrial systems of ethanol production operate under non-sterile conditions, microbial subpopulations are generated, depending on the operational conditions and the levels of contaminants. Among the most critical requirements for production of the second-generation ethanol is the reduction in the levels of toxic by-products of the lignocellulosic hydrolysates and the production of low-cost and efficient cellulosic enzymes. A number of procedures have been established for the conversion of lignocellulosic materials into ethanol, but none of them are completely satisfactory when process time, costs, and efficiency are considered.     

Development of minimal fermentation media supplementation for ethanol production using two Saccharomyces cerevisiae strains

Ethanol production by fermentation is strongly dependent on media composition. Specific nutrients, such as trace elements, vitamins and nitrogen will affect the physiological state and, consequently, the fermentation performance of the micro-organism employed. The purpose of this study has been to assess the highest ethanol production by a minimal medium, instead of the more complex nutrients supplementation used during alcoholic fermentation. All fermentation tests were carried out using a microwell plate reader to monitor the processes. Two Saccharomyces cerevisiae strains (NCYC 2826 and NCYC 3445) were tested using three nitrogen sources, supplied with different vitamin and salts. The results show that solutions made of urea phosphate, KCl, MgSO₄·7H₂O, Ca-panthothenate, biotin allowed an ethanol yield of 22.9 and 23.4 g/L for strain NCYC 2826 and NCYC 3445, respectively, representing 90 and 92% of the theoretical yield. All tests were carried out using glucose as common reference carbon source.     

Mathematical modeling of cellulose conversion to ethanol by the simultaneous saccharification and fermentation process

Ethanol, a promising alternative fuel, can be produced by the simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass, which combines the enzymatic hydrolysis of cellulose to glucose and the fermentation of glucose to ethanol by yeast in a single step.

A mathematical model that depicts the kinetics of SSF has been developed based on considerations of the quality of the substrate and enzyme, and the substrate-enzyme-microorganism interactions. Critical experimentation has been performed in conjunction with multiresponse nonlinear regression analysis to determine key model parameters regarding cell growth and ethanol production. The model will be used for rational SSF optimization and scale-up.

     

Ethanol production using concentrated oak wood hydrolysates and methods to detoxify

Ethanol production from concentrated oak wood hydrolysate was carried out to obtain a high ethanol concentration and a high ethanol yield. The effect of added inhibitory compounds, which are typically produced in the pretreatment step of steam-explosion on ethanol fermentation, was also examined. p-Hydroxybenzoic aldehyde, a lignin-degradation product, was the most inhibitory compound tested in this study. Compounds with additional methyl groups had reduced toxicity and the aromatic acids were less toxic than the corresponding aldehydes. The lignin-degradation products were more inhibitory than the sugar-derived products, such as furfural and 5-hydroxymethylfurfural (HMF). Adaptation of yeast cells to the wood hydrolysate and detoxification methods, such as using charcoal and overlime, had some beneficial effects on ethanol production using the concentrated wood hydrolysate. After treatment with charcoal and low-temperature sterilization, the yeast cells could utilize the concentrated wood hydrolysate with 170 as well as 140 g/L glucose, and produce 69.9 and 74.2 g/L ethanol, respectively, with a yield of 0.46–0.48 g ethanol/g glucose. In contrast, the cells could not completely utilize untreated wood hydrolysate with 100 g/L glucose. Low-temperature sterilization, with or without charcoal treatment, was very effective for ethanol production when highly concentrated wood hydrolysates were used. Low-temperature sterilization has advantages over traditional detoxification methods, such as using overlime, ion exchange, and charcoal, because of the reduction in the total cost of ethanol production.     

Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates

The Department of Energy’s Office of the Biomass Program has set goals of making ethanol cost competitive by 2012 and replacing 30% of 2004 transportation supply with biofuels by 2030. Both goals require improvements in conversions of cellulosic biomass to sugars as well as improvements in fermentation rates and yields. Current best pretreatment processes are reasonably efficient at making the cellulose/hemicellulose/lignin matrix amenable to enzymatic hydrolysis and fermentation, but they release a number of toxic compounds into the hydrolysate which inhibit the growth and ethanol productivity of fermentation organisms. Conditioning methods designed to reduce the toxicity of hydrolysates are effective, but add to process costs and tend to reduce sugar yields, thus adding significantly to the final cost of production. Reducing the cost of cellulosic ethanol production will likely require enhanced understanding of the source and mode of action of hydrolysate toxic compounds, the means by which some organisms resist the actions of these compounds, and the methodology and mechanisms for conditioning hydrolysate to reduce toxicity. This review will provide an update on the state of knowledge in these areas and can provide insights useful for the crafting of hypotheses for improvements in pretreatment, conditioning, and fermentation organisms.     

The Operable Modeling of Simultaneous Saccharification and Fermentation of Ethanol Production from Cellulose

An operable batch model of simultaneous saccharification and fermentation (SSF) for ethanol production from cellulose has been developed. The model includes four ordinary differential equations that describe the changes of cellobiose, glucose, yeast, and ethanol concentrations with respect to time. These equations were used to simulate the experimental data of the four main components in the SSF process of ethanol production from microcrystalline cellulose (Avicel PH101). The model parameters at 95% confidence intervals were determined by a MATLAB program based on the batch experimental data of the SSF. Both experimental data and model simulations showed that the cell growth was the rate-controlling step at the initial period in a series of reactions of cellulose to ethanol, and later, the conversion of cellulose to cellobiose controlled the process. The batch model was extended to the continuous and fed-batch operating models. For the continuous operation in the SSF, the ethanol productivities increased with increasing dilution rate, until a maximum value was attained, and rapidly decreased as the dilution rate approached the washout point. The model also predicted a relatively high ethanol mass for the fed-batch operation than the batch operation.     

Element concentrations of dry-grind corn-processing streams

The dry-grind corn process is one of two technologies used to convert corn into ethanol. In his process, all kernel components are processed through several sequential steps, including fermentation. Only one coproduct (distillers’ dried grains with solubles [DDGS]) is available for marketing. DDGS provide income to offset costs of processing; issues that affect marketing have implications in the economic viability of dry-grind plants. Two issues relate to elements in DDGS: high concentrations and excessive variation. Data on element concentrations in dry-grind processing streams could be helpful in addressing these concerns. The objective of this study was to determine element concentrations in primary process streams from dry-grind plants. Samples of corn, ground corn, beer, wet grains, syrup, and DDGS were obtained from nine dry-grind plants, and element concentrations were determinined. The concentrations of most elements in corn were not different among processing plants and were similar to published data. However, for the processing streams, there were differences in several element concentrations among processing plants. The concentrations of most elements in beer were about three times those of corn, due to the disappearance of starch during fermentation. Syrup had the highest element concentrations. Variations in element contents of DDGS and parent streams were due to processing conditions and not corn. Appropriate processing of thin stillage (the parent stream of syrup) could reduce the element content of DDGS.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (m _e ) rather than the max growth yield coefficient (Y _x ^sάx ). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.     

Kinetics of ethanol production from carob pods extract by immobilizedSaccharomyces cerevisiae cells

Kinetics of ethanol production from carob pods extract by immobilizedS. cerevisiae cells in static and shake flask fermentation have been investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The optimum values of ethanol concentration, ethanol productivity, ethanol yield, and fermentation efficiency were obtained at pH range 3.5–6.5 and temperature between 30–35°C. A maximum ethanol concentration (65 g/L), ethanol productivity (8.3 g/Lh), ethanol yield (0.44 g/g), and fermentation efficiency (95%) was achieved at an initial sugar concentration of 200, 150, 100, and 200 g/L, respectively. The highest values of specific ethanol production rate and specific sugar uptake rate were obtained at pH 6.5, temperature 40°C, and initial sugar concentration of 100 g/L. Other kinetic parameters, biomass concentration, biomass yield, and specific biomass production rate were maximum at pH 5.5, temperature 30°C, and initial sugar concentration 150 g/L. Under the same fermentation conditions non-sterilized carob pod extract gave higher ethanol concentration than sterilized medium. In repeated batch fermentations, the immobilizedS. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 5 d.