Restructuring upstream bioprocessing: technological and economical aspects for production of a generic microbial feedstock from wheat

Restructuring and optimization of the conventional fermentation industry for fuel and chemical production is necessary to replace petrochemical production routes. Guided by this concept, a novel biorefinery process has been developed as an alternative to conventional upstream processing routes, leading to the production of a generic fermentation feedstock from wheat. The robustness of Aspergillus awamori as enzyme producer is exploited in a continuous fungal fermentation on whole wheat flour. Vital gluten is extracted as an added-value byproduct by the conventional Martin process from a fraction of the overall wheat used. Enzymatic hydrolysis of gluten-free flour by the enzyme complex produced by A. awamori during fermentation produces a liquid stream rich in glucose (320 g/L). Autolysis of fungal cells produces a micronutrient-rich solution similar to yeast extract (1.6 g/L nitrogen, 0.5 g/L phosphorus). The case-specific combination of these two liquid streams can provide a nutrient-complete fermentation medium for a spectrum of microbial bioconversions for the production of such chemicals as organic acids, amino acids, bioethanol, glycerol, solvents, and microbial biodegradable plastics. Preliminary economic analysis has shown that the operating cost required to produce the feedstock is dependent on the plant capacity, cereal market price, presence and market value of added-value byproducts, labor costs, and mode of processing (batch or continuous). Integration of this process in an existing fermentation plant could lead to the production of a generic feedstock at an operating cost lower than the market price of glucose syrup (90% to 99% glucose) in the EU, provided that the plant capacity exceeds 410 m(3)/day. Further process improvements are also suggested.     

Improving wheat flour hydrolysis by an enzyme mixture from solid state fungal fermentation

In traditional cereal-based industrial processes, component separation is often incomplete resulting in a residue of mixed macromolecules including largely starch, protein, phytic acid and many others. The development of a viable cereal-based biorefinery would involve effective bioconversion of cereal components for the production of a nutrient-complete fermentation feedstock. Simultaneous starch and protein hydrolysis represents an effective approach to the production of platform chemicals from wheat. Solid state fermentations of wheat pieces and waste bread by Aspergillus oryzae and Aspergillus awamori have been combined in this study to enhance starch and protein hydrolysis. Kinetic studies confirmed that the proteolytic enzymes from A. oryzae introduced no negative effect on the stability of the amylolytic enzymes from A. awamori under the optimal conditions for starch hydrolysis. When applied to hydrolyse wheat flour, the enzyme solution from A. awamori converted nearly all of the starch into glucose and 23% of the total nitrogen (TN) into free amino nitrogen (FAN). Under the same reaction conditions the enzyme solution from A. oryzae hydrolysed 38% of the protein but only 18.5% of the starch. A mixture of the two enzyme solutions hydrolysed 34.1% of the protein, a 1.5-fold increase from that achieved by the enzyme solution from A. awamori, while maintaining a near completion of starch hydrolysis.     

Ethanol production from non-starch carbohydrates of wheat bran

Wheat bran (WB), produced worldwide in large quantities as a by-product of the wheat milling industry, constitutes a significant underutilized source of sugars. This paper describes various methods of hydrolyzing the abundant polysaccharides in bran to yield a sugar feedstock suitable for fermentation into bioethanol. Firstly, the starch in the bran was released using amylolytic enzymes. The fibrous material remaining was further hydrolyzed. Acid hydrolysis, heat pretreatment followed by enzymatic hydrolysis and direct enzymatic hydrolysis were compared in terms of total sugar yield and pentose sugar yield. The maximum total sugar yield was achieved when small amounts of acid were added at the pretreatment step prior to enzymatic hydrolysis. This form of pretreatment released most pentosans and significantly enhanced the hydrolysis of cellulose. The overall sugar yield of this combined hydrolysis method reached 80% of the theoretical and it consisted of 13.5 g arabinose, 22.8 g xylose and 16.7 g glucose per 100 g starch-free bran.     

Production of bioethanol and biodiesel using instant noodle waste

Instant noodle manufacturing waste was used as feedstock to convert it into two products, bioethanol and biodiesel. The raw material was pretreated to separate it into two potential feedstocks, starch residues and palm oil, for conversion to bioethanol and biodiesel, respectively. For the production of bioethanol, starch residues were converted into glucose by α-amylase and glucoamylase. To investigate the saccharification process of the pretreated starch residues, the optimal pretreatment conditions were determined. The bioethanol conversion reached 98.5 % of the theoretical maximum by Saccharomyces cerevisiae K35 fermentation after saccharification under optimized pretreatment conditions. Moreover, palm oil, isolated from the instant noodle waste, was converted into valuable biodiesel by use of immobilized lipase (Novozym 435). The effects of four categories of alcohol, oil-to-methanol ratio, reaction time, lipase concentration and water content on the conversion process were investigated. The maximum biodiesel conversion was 95.4 %.     

Impact of protein concentrate coproducts on net land requirement for European biofuel production

A methodology is proposed for calculating the net land area requirement for European biofuels, accounting for the substitution impact of animal feed protein coproducts such as dried distillers grains and solubles (DDGS) and rape meal. For example, when bioethanol is produced from cereal grain starch, grain protein is preserved in the DDGS coproduct. Each tonne of wheat DDGS has the potential to replace 0.59 tonnes of soy meal and 0.39 tonnes of cereals in EU animal feed, and the land area required for soy and cereal feed production offsets much of the land requirement for wheat bioethanol feedstock. While the land area needed for bioethanol from feed wheat in North West Europe is 0.40 ha t^?1, the net requirement after accounting for coproducts is just 0.03ha t^?1 of bioethanol produced, 6% of the gross land requirement. Calculated in this way, the net land area required to produce biofuel from EU cereal, rapeseed and sugar beet crops is much lower than the gross land requirement, and from cereal and sugar beet crops is less than the land requirement of biofuel from oil palm and sugar cane.     

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Key message

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain.

Abstract

Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85 °C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 % (14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.     

Molecular aspects of sucrose transport and its metabolism to starch during seed development in wheat: A comprehensive review

Wheat is one of the most important crops globally, and its grain is mainly used for human food, accounting for 20% of the total dietary calories. It is also used as animal feed and as a raw material for a variety of non-food and non-feed industrial products such as a feedstock for the production of bioethanol. Starch is the major constituent of a wheat grain, as a result, it is considered as a critical determinant of wheat yield and quality. The amount and composition of starch deposited in wheat grains is controlled primarily by sucrose transport from source tissues to the grain and its conversion to starch. Therefore, elucidation of the molecular mechanisms regulating these physiological processes provides important opportunities to improve wheat starch yield and quality through biotechnological approaches. This review comprehensively discusses the current understanding of the molecular aspects of sucrose transport and sucrose-to-starch metabolism in wheat grains. It also highlights the advances and prospects of starch biotechnology in wheat.     

Applicability of near-infrared spectroscopy for process monitoring in bioethanol production

The applicability of near-infrared (NIR) spectroscopy to bioethanol production is investigated. The NIR technique can provide assistance for rapid process monitoring, because organic compounds absorb radiation in the wavelength range 1100–2300 nm. For quantification of a sample's chemical composition, a calibration model is required that relates the measured spectral NIR absorbances to concentrations. For calibration, the concentrations in g/l are determined by the analytical reference method high performance liquid chromatography (HPLC). The calibration models are built and validated for moisture, protein, and starch in the feedstock material, and for glucose, ethanol, glycerol, lactic acid, acetic acid, maltose, fructose, and arabinose in the processed broths. These broths are prepared in laboratory experiments: The ground cereal samples are fermented to alcoholic broths (‘mash’), which are divided into an ethanol fraction and the residual fraction ‘stillage’ by distillation. The NIR technology together with chemometrics proved itself beneficial for fast monitoring of the current state of the bioethanol process, primarily for higher concentrated substances (>1 g/l).     

Enhanced bioenergy recovery from rapeseed plant in a biorefinery concept

The present study investigated the utilization of the whole rapeseed plant (seed and straw) for multi-biofuels production in a biorefinery concept. Results showed that bioethanol production from straw was technically feasible with ethanol yield of 0.15 g ethanol/g dry straw after combined alkaline peroxide and stream pretreatment. The byproducts (rapeseed cake, glycerol, hydrolysate and stillage) were evaluated for hydrogen and methane production. In batch experiments, the energy yields from each feedstock for, either methane production alone or for both hydrogen and methane, were similar. However, results from continuous experiments demonstrated that the two-stage hydrogen and methane fermentation process could work stably at organic loading rate up to 4.5 gVS/(Ld), while the single-stage methane production process failed. The energy recovery efficiency from rapeseed plant increased from 20% in the conventional biodiesel process to 60% in the biorefinery concept, by utilization of the whole rapeseed plant for biodiesel, bioethanol, biohydrogen and methane production.     

Process design and optimization of bioethanol production from cassava bagasse using statistical design and genetic algorithm

Abstract

Bioethanol production from agro-industrial residues is gaining attention because of the limited production of starch grains and sugarcane, and food–fuel conflict. The aim of the present study is to maximize the bioethanol production using cassava bagasse as a feedstock. Enzymatic liquefaction, by α-amylase, followed by simultaneous saccharification and fermentation (SSF), using glucoamylase and Zymomonas mobilis MTCC 2427, was investigated for bioethanol production from cassava bagasse. The factors influencing ethanol production process were identified and screened for significant factors using Plackett–Burman design. The significant factors (cassava bagasse concentration (10–50?g/L), concentration of α-amylase (5–25% (v/v), and temperature of fermentation (27–37?°C)) were optimized by employing Box–Behnken design and genetic algorithm. The maximum ethanol concentrations of 25.594?g/L and 25.910?g/L were obtained from Box–Behnken design and genetic algorithm, respectively, under optimum conditions. Thus, the study provides valuable insights in utilizing the cost-effective industrial residue, cassava bagasse, for the bioethanol production.     

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.     

A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid

In this study, a novel generic feedstock production strategy based on solid-state fermentation (SSF) has been developed and applied to the fermentative production of succinic acid. Wheat was fractionated into bran, gluten and gluten-free flour by milling and gluten extraction processes. The bran, which would normally be a waste product of the wheat milling industry, was used to produce glucoamylase and protease enzymes via SSF using Aspergillus awamori and Aspergillus oryzae, respectively. The resulting solutions were separately utilised for the hydrolysis of gluten-free flour and gluten to generate a glucose-rich stream of over 140gl(-1) glucose and a nitrogen-rich stream of more than 3.5gl(-1) free amino nitrogen. A microbial feedstock consisting of these two streams contained all the essential nutrients required for succinic acid fermentations using Actinobacillus succinogenes. In a fermentation using only the combined hydrolysate streams, around 22gl(-1) succinic acid was produced. The addition of MgCO(3) into the wheat-derived medium improved the succinic acid production further to more than 64gl(-1). These results demonstrate the SSF-based strategy is a successful approach for the production of a generic feedstock from wheat, and that this feedstock can be efficiently utilised for succinic acid production.     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

Two-stage hydrolysis of invasive algal feedstock for ethanol fermentation

The overall goal of this work was to develop a saccharification method for the production of third generation biofuel (i.e. bioethanol) using feedstock of the invasive marine macroalga Gracilaria salicornia. Under optimum conditions (120 °C and 2% sulfuric acid for 30 min), dilute acid hydrolysis of the homogenized invasive plants yielded a low concentration of glucose (4.1 mM or 4.3 g glucose/kg fresh algal biomass). However, two-stage hydrolysis of the homogenates (combination of dilute acid hydrolysis with enzymatic hydrolysis) produced 13.8 g of glucose from one kilogram of fresh algal feedstock. Batch fermentation analysis produced 79.1 g EtOH from one kilogram of dried invasive algal feedstock using the ethanologenic strain Escherichia coli KO11. Furthermore, ethanol production kinetics indicated that the invasive algal feedstock contained different types of sugar, including C(5) -sugar. This study represents the first report on third generation biofuel production from invasive macroalgae, suggesting that there is great potential for the production of renewable energy using marine invasive biomass.     

微生物在生物能源生产中的应用(英文)

生物可再生能源是最有前景的石油替代品之一.生物能源的生产原料包括:植物、有机废弃物和微生物.微生物在生物能源生产上有着广泛的应用,利用微生物制备的主要生物能源包括:生物柴油、生物乙醇、生物甲烷等.某些微生物如微藻和真菌可以生产大量油脂,这些油脂可以转化为生物柴油;有些微生物如酵母可以将糖类、淀粉以及纤维素转化为燃料乙醇,添加乙醇的汽油或柴油燃烧排放明显降低;还有些厌氧微生物可以将有机废弃物转化为甲烷,可用做家用燃气、车用燃气或发电.除此之外微生物还具有在生产能源的同时治理环境污染的优势.总之研究开发微生物在生物能源生产中的应用有利于世界可持续发展.     

Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases

Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.     

Dilute acid pretreatment and enzymatic saccharification of sugarcane tops for bioethanol production

The aim of this work was to study the feasibility of using sugarcane tops as feedstock for the production of bioethanol. The process involved the pretreatment using acid followed by enzymatic saccharification using cellulases and the process was optimized for various parameters such as biomass loading, enzyme loading, surfactant concentration and incubation time using Box–Behnken design. Under optimum hydrolysis conditions, 0.685 g/g of reducing sugar was produced per gram of pretreated biomass. The fermentation of the hydrolyzate using Saccharomyces cerevisae produced 11.365 g/L of bioethanol with an efficiency of about 50%. This is the first report on utilization of sugarcane tops for bioethanol production.     

Process design and optimization of novel wheat-based continuous bioethanol production system

A novel design of a wheat-based biorefinery for bioethanol production, including wheat milling, gluten extraction as byproduct, fungal submerged fermentation for enzyme production, starch hydrolysis, fungal biomass autolysis for nutrient regeneration, yeast fermentation with recycling integrated with a pervaporation membrane for ethanol concentration, and fuel-grade ethanol purification by pressure swing distillation (PSD), was optimized in continuous mode using the equation-based software General Algebraic Modelling System (GAMS). The novel wheat biorefining strategy could result in a production cost within the range of dollars 0.96-0.50 gal(-1) ethanol (dollars 0.25-0.13 L(-1) ethanol) when the production capacity of the plant is within the range of 10-33.5 million gal y(-1) (37.85-126.8 million L y(-1)). The production of value-added byproducts (e.g., bran-rich pearlings, gluten, pure yeast cells) was identified as a crucial factor for improving the economics of fuel ethanol production from wheat. Integration of yeast fermentation with pervaporation membrane could result in the concentration of ethanol in the fermentation outlet stream (up to 40 mol %). The application of a PSD system that consisted of a low-pressure and a high-pressure column and employing heat integration between the high- and low-pressure columns resulted in reduced operating cost (up to 44%) for fuel-grade ethanol production.     

Co-fermentation of carbon sources by Enterobacter aerogenes ATCC 29007 to enhance the production of bioethanol

We investigated the enhancement of bioethanol production in Enterobacter aerogenes ATCC 29007 by co-fermentation of carbon sources such as glycerol, glucose, galactose, sucrose, fructose, xylose, starch, mannitol and citric acid. Biofuel production increases with increasing growth rate of microorganisms; that is why we investigated the optimal growth rate of E. aerogenes ATCC 29007, using mixtures of different carbon sources with glycerol. E. aerogenes ATCC 29007 was incubated in media containing each carbon source and glycerol; growth rate and bioethanol production improved in all cases compared to those in medium containing glycerol alone. The growth rate and bioethanol production were highest with mannitol. Fermentation was carried out at 37 °C for 18 h, pH 7, using 50 mL defined production medium in 100 mL serum bottles at 200 rpm. Bioethanol production under optimized conditions in medium containing 16 g/L mannitol and 20 g/L glycerol increased sixfold (32.10 g/L) than that containing glycerol alone (5.23 g/L) as the carbon source in anaerobic conditions. Similarly, bioethanol production using free cells in continuous co-fermentation also improved (27.28 g/L) when 90.37 % of 16 g/L mannitol and 67.15 % of 20 g/L glycerol were used. Although naturally existing or engineered microorganisms can ferment mixed sugars sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Here, we present new findings in E. aerogenes ATCC 29007 that can be used to improve bioethanol production by simultaneous co-fermentation of glycerol and mannitol.     

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75?% of the theoretical maximum yield.