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1.  Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility 
Indian Journal of Microbiology  2011;52(2):122-130.
Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.
PMCID: PMC3386436  PMID: 23729871
Bioethanol; Biological pre-treatment; Delignification; Lignocellulose
2.  Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture 
Lignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity).
Ethanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana “protect” C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively.
This work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.
PMCID: PMC3653780  PMID: 23628342
Consortia; Consolidated bioprocessing; Cellulosic ethanol; Symbiosis; Oxygen transport
3.  Land usage attributed to corn ethanol production in the United States: sensitivity to technological advances in corn grain yield, ethanol conversion, and co-product utilization 
Although the system for producing yellow corn grain is well established in the US, its role among other biofeedstock alternatives to petroleum-based energy sources has to be balanced with its predominant purpose for food and feed as well as economics, land use, and environmental stewardship. We model land usage attributed to corn ethanol production in the US to evaluate the effects of anticipated technological change in corn grain production, ethanol processing, and livestock feeding through a multi-disciplinary approach. Seven scenarios are evaluated: four considering the impact of technological advances on corn grain production, two focused on improved efficiencies in ethanol processing, and one reflecting greater use of ethanol co-products (that is, distillers dried grains with solubles) in diets for dairy cattle, pigs, and poultry. For each scenario, land area attributed to corn ethanol production is estimated for three time horizons: 2011 (current), the time period at which the 15 billion gallon cap for corn ethanol as per the Renewable Fuel Standard is achieved, and 2026 (15 years out).
Although 40.5% of corn grain was channeled to ethanol processing in 2011, only 25% of US corn acreage was attributable to ethanol when accounting for feed co-product utilization. By 2026, land area attributed to corn ethanol production is reduced to 11% to 19% depending on the corn grain yield level associated with the four corn production scenarios, considering oil replacement associated with the soybean meal substituted in livestock diets with distillers dried grains with solubles. Efficiencies in ethanol processing, although producing more ethanol per bushel of processed corn, result in less co-products and therefore less offset of corn acreage. Shifting the use of distillers dried grains with solubles in feed to dairy cattle, pigs, and poultry substantially reduces land area attributed to corn ethanol production. However, because distillers dried grains with solubles substitutes at a higher rate for soybean meal, oil replacement requirements intensify and positively feedback to elevate estimates of land usage.
Accounting for anticipated technological changes in the corn ethanol system is important for understanding the associated land base ascribed, and may aid in calibrating parameters for land use models in biofuel life-cycle analyses.
PMCID: PMC4022103  PMID: 24725504
Agricultural biotechnology; Corn ethanol; Corn ethanol co-products; Corn gluten feed; Corn gluten meal; Corn grain production; DDGS; Distillers dried grains with solubles; Livestock feeding; Technological change
4.  Simultaneous saccharification and cofermentation of lignocellulosic residues from commercial furfural production and corn kernels using different nutrient media 
As the supply of starch grain and sugar cane, currently the main feedstocks for bioethanol production, become limited, lignocelluloses will be sought as alternative materials for bioethanol production. Production of cellulosic ethanol is still cost-inefficient because of the low final ethanol concentration and the addition of nutrients. We report the use of simultaneous saccharification and cofermentation (SSCF) of lignocellulosic residues from commercial furfural production (furfural residue, FR) and corn kernels to compare different nutritional media. The final ethanol concentration, yield, number of live yeast cells, and yeast-cell death ratio were investigated to evaluate the effectiveness of integrating cellulosic and starch ethanol.
Both the ethanol yield and number of live yeast cells increased with increasing corn-kernel concentration, whereas the yeast-cell death ratio decreased in SSCF of FR and corn kernels. An ethanol concentration of 73.1 g/L at 120 h, which corresponded to a 101.1% ethanol yield based on FR cellulose and corn starch, was obtained in SSCF of 7.5% FR and 14.5% corn kernels with mineral-salt medium. SSCF could simultaneously convert cellulose into ethanol from both corn kernels and FR, and SSCF ethanol yield was similar between the organic and mineral-salt media.
Starch ethanol promotes cellulosic ethanol by providing important nutrients for fermentative organisms, and in turn cellulosic ethanol promotes starch ethanol by providing cellulosic enzymes that convert the cellulosic polysaccharides in starch materials into additional ethanol. It is feasible to produce ethanol in SSCF of FR and corn kernels with mineral-salt medium. It would be cost-efficient to produce ethanol in SSCF of high concentrations of water-insoluble solids of lignocellulosic materials and corn kernels. Compared with prehydrolysis and fed-batch strategy using lignocellulosic materials, addition of starch hydrolysates to cellulosic ethanol production is a more suitable method to improve the final ethanol concentration.
PMCID: PMC3161845  PMID: 21801455
5.  Utilization of household food waste for the production of ethanol at high dry material content 
Environmental issues and shortage of fossil fuels have turned the public interest to the utilization of renewable, environmentally friendly fuels, such as ethanol. In order to minimize the competition between fuels and food production, researchers are focusing their efforts to the utilization of wastes and by-products as raw materials for the production of ethanol. household food wastes are being produced in great quantities in European Union and their handling can be a challenge. Moreover, their disposal can cause severe environmental issues (for example emission of greenhouse gasses). On the other hand, they contain significant amounts of sugars (both soluble and insoluble) and they can be used as raw material for the production of ethanol.
Household food wastes were utilized as raw material for the production of ethanol at high dry material consistencies. A distinct liquefaction/saccharification step has been included to the process, which rapidly reduced the viscosity of the high solid content substrate, resulting in better mixing of the fermenting microorganism. This step had a positive effect in both ethanol production and productivity, leading to a significant increase in both values, which was up to 40.81% and 4.46 fold, respectively. Remaining solids (residue) after fermentation at 45% w/v dry material (which contained also the unhydrolyzed fraction of cellulose), were subjected to a hydrothermal pretreatment in order to be utilized as raw material for a subsequent ethanol fermentation. This led to an increase of 13.16% in the ethanol production levels achieving a final ethanol yield of 107.58 g/kg dry material.
In conclusion, the ability of utilizing household food waste for the production of ethanol at elevated dry material content has been demonstrated. A separate liquefaction/saccharification process can increase both ethanol production and productivity. Finally, subsequent fermentation of the remaining solids could lead to an increase of the overall ethanol production yield.
PMCID: PMC3892076  PMID: 24401142
Ethanol; Liquefaction; Saccharification; Household food waste; Residue solids; Subsequent fermentation; Saccharomyces cerevisiae
6.  Quantifying the metabolic capabilities of engineered Zymomonas mobilis using linear programming analysis 
The need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, "clean" methods for their conversion into higher fuels becomes imperative. Ethanol, whose significance as fuel has dramatically increased in the last decade, can be produced from hexoses and pentoses through microbial fermentation. Importantly, plant biomass, if appropriately and effectively decomposed, is a potential inexpensive and highly renewable source of the hexose and pentose mixture. Recently, the engineered (to also catabolize pentoses) anaerobic bacterium Zymomonas mobilis has been widely discussed among the most promising microorganisms for the microbial production of ethanol fuel. However, Z. mobilis genome having been fully sequenced in 2005, there is still a small number of published studies of its in vivo physiology and limited use of the metabolic engineering experimental and computational toolboxes to understand its metabolic pathway interconnectivity and regulation towards the optimization of its hexose and pentose fermentation into ethanol.
In this paper, we reconstructed the metabolic network of the engineered Z. mobilis to a level that it could be modelled using the metabolic engineering methodologies. We then used linear programming (LP) analysis and identified the Z. mobilis metabolic boundaries with respect to various biological objectives, these boundaries being determined only by Z. mobilis network's stoichiometric connectivity. This study revealed the essential for bacterial growth reactions and elucidated the association between the metabolic pathways, especially regarding main product and byproduct formation. More specifically, the study indicated that ethanol and biomass production depend directly on anaerobic respiration stoichiometry and activity. Thus, enhanced understanding and improved means for analyzing anaerobic respiration and redox potential in vivo are needed to yield further conclusions for potential genetic targets that may lead to optimized Z. mobilis strains.
Applying LP to study the Z. mobilis physiology enabled the identification of the main factors influencing the accomplishment of certain biological objectives due to metabolic network connectivity only. This first-level metabolic analysis model forms the basis for the incorporation of more complex regulatory mechanisms and the formation of more realistic models for the accurate simulation of the in vivo Z. mobilis physiology.
PMCID: PMC1831482  PMID: 17349037
7.  How can accelerated development of bioenergy contribute to the future UK energy mix? Insights from a MARKAL modelling exercise 
This work explores the potential contribution of bioenergy technologies to 60% and 80% carbon reductions in the UK energy system by 2050, by outlining the potential for accelerated technological development of bioenergy chains. The investigation was based on insights from MARKAL modelling, detailed literature reviews and expert consultations. Due to the number and complexity of bioenergy pathways and technologies in the model, three chains and two underpinning technologies were selected for detailed investigation: (1) lignocellulosic hydrolysis for the production of bioethanol, (2) gasification technologies for heat and power, (3) fast pyrolysis of biomass for bio-oil production, (4) biotechnological advances for second generation bioenergy crops, and (5) the development of agro-machinery for growing and harvesting bioenergy crops. Detailed literature searches and expert consultations (looking inter alia at research and development needs and economic projections) led to the development of an 'accelerated' dataset of modelling parameters for each of the selected bioenergy pathways, which were included in five different scenario runs with UK-MARKAL (MED). The results of the 'accelerated runs' were compared with a low-carbon (LC-Core) scenario, which assesses the cheapest way to decarbonise the energy sector.
Bioenergy was deployed in larger quantities in the bioenergy accelerated technological development scenario compared with the LC-Core scenario. In the electricity sector, solid biomass was highly utilised for energy crop gasification, displacing some deployment of wind power, and nuclear and marine to a lesser extent. Solid biomass was also deployed for heat in the residential sector from 2040 in much higher quantities in the bioenergy accelerated technological development scenario compared with LC-Core. Although lignocellulosic ethanol increased, overall ethanol decreased in the transport sector in the bioenergy accelerated technological development scenario due to a reduction in ethanol produced from wheat.
There is much potential for future deployment of bioenergy technologies to decarbonise the energy sector. However, future deployment is dependent on many different factors including investment and efforts towards research and development needs, carbon reduction targets and the ability to compete with other low carbon technologies as they become deployed. All bioenergy technologies should become increasingly more economically competitive with fossil-based technologies as feedstock costs and flexibility are reduced in line with technological advances.
PMCID: PMC2713217  PMID: 19575786
8.  Bioconversion of xylose, hexoses and biomass to ethanol by a new isolate of the white rot basidiomycete Trametes versicolor 
SpringerPlus  2014;3:121.
Second-generation bioethanol production requires the development of economically feasible and sustainable processes that use renewable lignocellulosic biomass as a starting material. However, the microbial fermentation of xylose, which is the principal pentose sugar in hemicellulose, is a limiting factor in developing such processes. Here, a strain of the white rot basidiomycete Trametes versicolor that was capable of efficiently fermenting xylose was newly isolated and characterized. This strain, designated KT9427, was capable of assimilating and converting xylose to ethanol under anaerobic conditions with a yield of 0.44 g ethanol per 1 g of sugar consumed. In culture medium containing low yeast extract concentrations, xylose consumption and ethanol productivity were enhanced. Adjusting the initial pH between 3.0 and 5.0 did not markedly influence xylose fermentation. T. versicolor KT9427 also produced ethanol from glucose, mannose, fructose, cellobiose and maltose at yields ranging from 0.45 to 0.49 g ethanol per 1 g of sugar consumed. In addition, strain KT9427 exhibited favourable conversion of non-pretreated starch, cellulose, xylan, wheat bran and rice straw into ethanol compared to common recombinant yeast strains. Taken together, the present findings suggest that T. versicolor KT9427 is a promising candidate for environmentally friendly ethanol production directly from lignocellulosic biomass.
PMCID: PMC3950376  PMID: 24624317
Xylose; Biomass; Ethanol; Fermentation; White rot basidiomycete
9.  Metabolic Engineering of Microorganisms for the Production of Higher Alcohols 
mBio  2014;5(5):e01524-14.
Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols.
PMCID: PMC4173780  PMID: 25182323
10.  Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa 
Interface Focus  2011;1(2):196-211.
The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic ethanol production into existing bio-based industries also using thermochemical processes to optimize energy balances is discussed. Biofuels have played a pivotal yet suboptimal role in supplementing Africa's energy requirements in the past. Capitalizing on sub-Saharan Africa's total biomass potential and using second-generation technologies merit a fresh look at the potential role of bioethanol production towards developing a sustainable Africa while addressing food security, human needs and local wealth creation.
PMCID: PMC3262263  PMID: 22482027
cellulosic ethanol; consolidated bioprocessing; integrating bio-based industries; sustainable biofuels in Africa
11.  The greenhouse gas emissions performance of cellulosic ethanol supply chains in Europe 
Calculating the greenhouse gas savings that may be attributed to biofuels is problematic because production systems are inherently complex and methods used to quantify savings are subjective. Differing approaches and interpretations have fuelled a debate about the environmental merit of biofuels, and consequently about the level of policy support that can be justified. This paper estimates and compares emissions from plausible supply chains for lignocellulosic ethanol production, exemplified using data specific to the UK and Sweden. The common elements that give rise to the greatest greenhouse gas emissions are identified and the sensitivity of total emissions to variations in these elements is estimated. The implications of including consequential impacts including indirect land-use change, and the effects of selecting alternative allocation methods on the interpretation of results are discussed.
We find that the most important factors affecting supply chain emissions are the emissions embodied in biomass production, the use of electricity in the conversion process and potentially consequential impacts: indirect land-use change and fertiliser replacement. The large quantity of electricity consumed during enzyme manufacture suggests that enzymatic conversion processes may give rise to greater greenhouse gas emissions than the dilute acid conversion process, even though the dilute acid process has a somewhat lower ethanol yield.
The lignocellulosic ethanol supply chains considered here all lead to greenhouse gas savings relative to gasoline An important caveat to this is that if lignocellulosic ethanol production uses feedstocks that lead to indirect land-use change, or other significant consequential impacts, the benefit may be greatly reduced.
Co-locating ethanol, electricity generation and enzyme production in a single facility may improve performance, particularly if this allows the number of energy intensive steps in enzyme production to be reduced, or if other process synergies are available. If biofuels policy in the EU remains contingent on favourable environmental performance then the multi-scale nature of bioenergy supply chains presents a genuine challenge. Lignocellulosic ethanol holds promise for emission reductions, but maximising greenhouse gas savings will not only require efficient supply chain design but also a better understanding of the spatial and temporal factors which affect overall performance.
PMCID: PMC2746206  PMID: 19682352
12.  Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production 
Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed.
To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.
Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.
PMCID: PMC3599373  PMID: 23374631
Cellulosic ethanol; Crystalline cellulose; Cocktail formulation; Synthetic biology; Consolidated bioprocess
13.  Bioconversion of Sugarcane Biomass into Ethanol: An Overview about Composition, Pretreatment Methods, Detoxification of Hydrolysates, Enzymatic Saccharification, and Ethanol Fermentation 
Depleted supplies of fossil fuel, regular price hikes of gasoline, and environmental damage have necessitated the search for economic and eco-benign alternative of gasoline. Ethanol is produced from food/feed-based substrates (grains, sugars, and molasses), and its application as an energy source does not seem fit for long term due to the increasing fuel, food, feed, and other needs. These concerns have enforced to explore the alternative means of cost competitive and sustainable supply of biofuel. Sugarcane residues, sugarcane bagasse (SB), and straw (SS) could be the ideal feedstock for the second-generation (2G) ethanol production. These raw materials are rich in carbohydrates and renewable and do not compete with food/feed demands. However, the efficient bioconversion of SB/SS (efficient pretreatment technology, depolymerization of cellulose, and fermentation of released sugars) remains challenging to commercialize the cellulosic ethanol. Among the technological challenges, robust pretreatment and development of efficient bioconversion process (implicating suitable ethanol producing strains converting pentose and hexose sugars) have a key role to play. This paper aims to review the compositional profile of SB and SS, pretreatment methods of cane biomass, detoxification methods for the purification of hydrolysates, enzymatic hydrolysis, and the fermentation of released sugars for ethanol production.
PMCID: PMC3516358  PMID: 23251086
14.  Solving ethanol production problems with genetically modified yeast strains 
Brazilian Journal of Microbiology  2014;44(3):665-671.
The current world demand for bioethanol is increasing as a consequence of low fossil fuel availability and a growing number of ethanol/gasoline flex-fuel cars. In addition, countries in several parts of the world have agreed to reduce carbon dioxide emissions, and the use of ethanol as a fuel (which produces fewer pollutants than petroleum products) has been considered to be a good alternative to petroleum products. The ethanol that is produced in Brazil from the first-generation process is optimized and can be accomplished at low cost. However, because of the large volume of ethanol that is produced and traded each year, any small improvement in the process could represent a savings of billions dollars. Several Brazilian research programs are investing in sugarcane improvement, but little attention has been given to the improvement of yeast strains that participate in the first-generation process at present. The Brazilian ethanol production process uses sugarcane as a carbon source for the yeast Saccharomyces cerevisiae. Yeast is then grown at a high cellular density and high temperatures in large-capacity open tanks with cells recycle. All of these culture conditions compel the yeast to cope with several types of stress. Among the main stressors are high temperatures and high ethanol concentrations inside the fermentation tanks during alcohol production. Moreover, the competition between the desired yeast strains, which are inoculated at the beginning of the process, with contaminants such as wild type yeasts and bacteria, requires acid treatment to successfully recycle the cells. This review is focused on describing the problems and stressors within the Brazilian ethanol production system. It also highlights some genetic modifications that can help to circumvent these difficulties in yeast.
PMCID: PMC3910172  PMID: 24516432
Brazilian ethanol production; yeast improvement; resistance to stress
15.  Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria 
Consolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.
We have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.
The newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.
PMCID: PMC3598825  PMID: 23448304
Anaerobic; Caldicellulosiruptor; Consolidated bioprocessing; Ethanol; Extremely thermophilic bacteria; High temperature; Lactate; Lignocellulose; Thermoanaerobacter
16.  One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae 
While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor.
Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8–12 FPU/ml throughout the one-pot process. When 50–300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7–46.3 g/l and 0.15–0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50–300 g SF/l, the ethanol concentration and yield were 9.5–35.1 g/l with their yields of 0.12–0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol.
A. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass.
PMCID: PMC3493283  PMID: 22938388
Bioethanol; Cellulase; Biomass; Acremonium cellulolyticus C-1; Saccharomyces cerevisiae; Biorefinery
17.  Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress 
BMC Genomics  2012;13:336.
Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis.
In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume.
This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.
PMCID: PMC3478167  PMID: 22823947
18.  Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol 
During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs.
The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses.
Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.
PMCID: PMC3986927  PMID: 24670111
Saccharomyces spp.; Phenotypic microarray; Bioethanol; Fermentation
19.  SSF of steam-pretreated wheat straw with the addition of saccharified or fermented wheat meal in integrated bioethanol production 
Integration of second-generation (2G) bioethanol production with existing first-generation (1G) production may facilitate commercial production of ethanol from cellulosic material. Since 2G hydrolysates have a low sugar concentration and 1G streams often have to be diluted prior to fermentation, mixing of streams is beneficial. Improved ethanol concentrations in the 2G production process lowers energy demand in distillation, improves overall energy efficiency and thus lower production cost. There is also a potential to reach higher ethanol yields, which is required in economically feasible ethanol production. Integrated process scenarios with addition of saccharified wheat meal (SWM) or fermented wheat meal (FWM) were investigated in simultaneous saccharification and (co-)fermentation (SSF or SSCF) of steam-pretreated wheat straw, while the possibility of recovering the valuable protein-rich fibre residue from the wheat was also studied.
The addition of SWM to SSF of steam-pretreated wheat straw, using commercially used dried baker’s yeast, S. cerevisiae, resulted in ethanol concentrations of about 60 g/L, equivalent to ethanol yields of about 90% of the theoretical. The addition of FWM in batch mode SSF was toxic to baker’s yeast, due to the ethanol content of FWM, resulting in a very low yield and high accumulation of glucose. The addition of FWM in fed-batch mode still caused a slight accumulation of glucose, but the ethanol concentration was fairly high, 51.2 g/L, corresponding to an ethanol yield of 90%, based on the amount of glucose added.
In batch mode of SSCF using the xylose-fermenting, genetically modified S. cerevisiae strain KE6-12, no improvement was observed in ethanol yield or concentration, compared with baker’s yeast, despite the increased xylose utilization, probably due to the considerable increase in glycerol production. A slight increase in xylose consumption was seen when glucose from SWM was fed at a low feed rate, after 48 hours, compared with batch SSCF. However, the ethanol yield and concentration remained in the same range as in batch mode.
Ethanol concentrations of about 6% (w/v) were obtained, which will result in a significant reduction in the cost of downstream processing, compared with SSF of the lignocellulosic substrate alone. As an additional benefit, it is also possible to recover the protein-rich residue from the SWM in the process configurations presented, providing a valuable co-product.
PMCID: PMC4176987  PMID: 24286350
SSF; SSCF; Wheat meal hydrolysate; Fermented wheat meal; Steam-pretreated wheat straw; Glucose and xylose co-fermentation
20.  The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass 
Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.
PMCID: PMC2726447  PMID: 19680472
bacteria; bioconversion; cellulases; cellulosome
21.  The commercial performance of cellulosic ethanol supply-chains in Europe 
The production of fuel-grade ethanol from lignocellulosic biomass resources has the potential to increase biofuel production capacity whilst minimising the negative environmental impacts. These benefits will only be realised if lignocellulosic ethanol production can compete on price with conventional fossil fuels and if it can be produced commercially at scale. This paper focuses on lignocellulosic ethanol production in Europe. The hypothesis is that the eventual cost of production will be determined not only by the performance of the conversion process but by the performance of the entire supply-chain from feedstock production to consumption. To test this, a model for supply-chain cost comparison is developed, the components of representative ethanol supply-chains are described, the factors that are most important in determining the cost and profitability of ethanol production are identified, and a detailed sensitivity analysis is conducted.
The most important cost determinants are the cost of feedstocks, primarily determined by location and existing markets, and the value obtained for ethanol, primarily determined by the oil price and policy incentives. Both of these factors are highly uncertain. The best performing chains (ethanol produced from softwood and sold as a low percentage blend with gasoline) could ultimately be cost competitive with gasoline without requiring subsidy, but production from straw would generally be less competitive.
Supply-chain design will play a critical role in determining commercial viability. The importance of feedstock supply highlights the need for location-specific assessments of feedstock availability and price. Similarly, the role of subsidies and policy incentives in creating and sustaining the ethanol market highlights the importance of political engagement and the need to include political risks in investment appraisal. For the supply-chains described here, and with the cost and market parameters selected, selling ethanol as a low percentage blend with gasoline will maximise ethanol revenues and minimise the need for subsidies. It follows, therefore, that the market for low percentage blends should be saturated before markets for high percentage blends.
PMCID: PMC2654549  PMID: 19192294
22.  A constraint-based model of Scheffersomyces stipitis for improved ethanol production 
As one of the best xylose utilization microorganisms, Scheffersomyces stipitis exhibits great potential for the efficient lignocellulosic biomass fermentation. Therefore, a comprehensive understanding of its unique physiological and metabolic characteristics is required to further improve its performance on cellulosic ethanol production.
A constraint-based genome-scale metabolic model for S. stipitis CBS 6054 was developed on the basis of its genomic, transcriptomic and literature information. The model iTL885 consists of 885 genes, 870 metabolites, and 1240 reactions. During the reconstruction process, 36 putative sugar transporters were reannotated and the metabolisms of 7 sugars were illuminated. Essentiality study was conducted to predict essential genes on different growth media. Key factors affecting cell growth and ethanol formation were investigated by the use of constraint-based analysis. Furthermore, the uptake systems and metabolic routes of xylose were elucidated, and the optimization strategies for the overproduction of ethanol were proposed from both genetic and environmental perspectives.
Systems biology modelling has proven to be a powerful tool for targeting metabolic changes. Thus, this systematic investigation of the metabolism of S. stipitis could be used as a starting point for future experiment designs aimed at identifying the metabolic bottlenecks of this important yeast.
PMCID: PMC3503688  PMID: 22998943
Scheffersomyces stipitis; Genome-scale metabolic model; Constraint-based simulation; Xylose utilization; Ethanol production
23.  Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria 
BMC Microbiology  2012;12:295.
Fermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae.
Bioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do.
Here we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.
PMCID: PMC3561251  PMID: 23249097
24.  Aerobic and sequential anaerobic fermentation to produce xylitol and ethanol using non-detoxified acid pretreated corncob 
Biotechnology for Biofuels  2014;7(1):166.
For economical bioethanol production from lignocellulosic materials, the major technical challenges to lower the production cost are as follows: (1) The microorganism should use efficiently all glucose and xylose in the lignocellulose hydrolysate. (2) The microorganism should have high tolerance to the inhibitors present in the lignocellulose hydrolysate. The aim of the present work was to combine inhibitor degradation, xylitol fermentation, and ethanol production using a single yeast strain.
A new process of integrated aerobic xylitol production and anaerobic ethanol fermentation using non-detoxified acid pretreated corncob by Candida tropicalis W103 was proposed. C. tropicalis W103 is able to degrade acetate, furfural, and 5-hydromethylfurfural and metabolite xylose to xylitol under aerobic conditions, and the aerobic fermentation residue was used as the substrate for ethanol production by anaerobic simultaneous saccharification and fermentation. With 20% substrate loading, furfural and 5-hydroxymethylfurfural were degraded totally after 60 h aerobic incubation. A maximal xylitol concentration of 17.1 g l-1 was obtained with a yield of 0.32 g g-1 xylose. Then under anaerobic conditions with the addition of cellulase, 25.3 g l-1 ethanol was produced after 72 h anaerobic fermentation, corresponding to 82% of the theoretical yield.
Xylitol and ethanol were produced in Candida tropicalis W103 using dual-phase fermentations, which comprise a changing from aerobic conditions (inhibitor degradation and xylitol production) to anaerobic simultaneous saccharification and ethanol fermentation. This is the first report of integrated xylitol and ethanol production from non-detoxified acid pretreated corncob using a single microorganism.
PMCID: PMC4245779  PMID: 25431622
Bioconversion; Corncob; Fermentation; Microbial Growth; Inhibitor
25.  Simultaneous saccharification and co-fermentation for bioethanol production using corncobs at lab, PDU and demo scales 
While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising process for bioconversion of lignocellulosic materials to ethanol, there are still relatively little demo-plant data and operating experiences reported in the literature. In the current work, we designed a SSCF process and scaled up from lab to demo scale reaching 4% (w/v) ethanol using xylose rich corncobs.
Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluated for their fermentation performance in hydrolysates of steam pretreated corncobs. Two strains, RHD-15 and KE6-12 with highest ethanol yield and lowest xylitol yield, respectively were further screened in SSCF using the whole slurry from pretreatment. Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the preferred strain since it produced 26% lower xylitol from consumed xylose compared to RHD-15. Model SSCF experiments with glucose or hydrolysate feed in combination with prefermentation resulted in 79% of xylose consumption and more than 75% of the theoretical ethanol yield on available glucose and xylose in lab and PDU scales. The results suggest that for an efficient xylose conversion to ethanol controlled release of glucose from enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout the SSCF. Fed-batch SSCF in PDU with addition of enzymes at three different time points facilitated controlled release of glucose and hence co-consumption of glucose and xylose was observed yielding 76% of the theoretical ethanol yield on available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF in combination with prefermentation and a feed of substrate and enzymes 47 and 40 g l-1 of ethanol corresponding to 68% and 58% of the theoretical ethanol yield on available glucose and xylose were produced at 10.5% WIS in PDU and demo scale, respectively. The strain KE6-12 was able to completely consume xylose within 76 h during the fermentation of hydrolysate in a 10 m3 demo scale bioreactor.
The potential of SSCF is improved in combination with prefermentation and a feed of substrate and enzymes. It was possible to successfully reproduce the fed-batch SSCF at demo scale producing 4% (w/v) ethanol which is the minimum economical requirement for efficient lignocellulosic bioethanol production process.
PMCID: PMC3598390  PMID: 23311728
S. cerevisiae; SSCF; Prefermentation; Xylose

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