Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing

With the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundant lignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractive option. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a very rigid and complex structure. It is accordingly much more expensive to process than starchy grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose are required. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme production, saccharification, and fermentation, is considered a promising strategy for reducing production costs.     

Plant genetic engineering to improve biomass characteristics for biofuels

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.     

Bioprocessing for biofuels

While engineering of new biofuels pathways into microbial hosts has received considerable attention, innovations in bioprocessing are required for commercialization of both conventional and next-generation fuels. For ethanol and butanol, reducing energy costs for product recovery remains a challenge. Fuels produced from heterologous aerobic pathways in yeast and bacteria require control of aeration and cooling at large scales. Converting lignocellulosic biomass to sugars for fuels production requires effective biomass pretreatment to increase surface area, decrystallize cellulose and facilitate enzymatic hydrolysis. Effective means to recover microalgae and extract their intracellular lipids remains a practical and economic bottleneck in algal biodiesel production.     

Biomass deconstruction to sugars

The production of biofuels from lignocellulosic biomass relies on the depolymerization of its polysaccharide content into fermentable sugars. Accomplishing this requires pretreatment of the biomass to reduce its size, and chemical or physical alteration of the biomass polymers to enhance the susceptibility of their glycosidic linkages to enzymatic or acid catalyzed cleavage. Well-studied approaches include dilute and concentrated acid pretreatment and catalysis, and the dissolution of biomass in organic solvents. These and recently developed approaches, such as solubilization in ionic liquids, are reviewed in terms of the chemical and physical changes occurring in biomass pretreatment. As pretreatment represents one of the major costs in converting biomass to fuels, the factors that contribute to pretreatments costs, and their impact on overall process economics, are described.     

Fungal pretreatment of lignocellulosic biomass

Pretreatment is a crucial step in the conversion of lignocellulosic biomass to fermentable sugars and biofuels. Compared to thermal/chemical pretreatment, fungal pretreatment reduces the recalcitrance of lignocellulosic biomass by lignin-degrading microorganisms and thus potentially provides an environmentally-friendly and energy-efficient pretreatment technology for biofuel production. This paper provides an overview of the current state of fungal pretreatment by white rot fungi for biofuel production. The specific topics discussed are: 1) enzymes involved in biodegradation during the fungal pretreatment; 2) operating parameters governing performance of the fungal pretreatment; 3) the effect of fungal pretreatment on enzymatic hydrolysis and ethanol production; 4) efforts for improving enzymatic hydrolysis and ethanol production through combinations of fungal pretreatment and physical/chemical pretreatment; 5) the treatment of lignocellulosic biomass with lignin-degrading enzymes isolated from fungal pretreatment, with a comparison to fungal pretreatment; 6) modeling, reactor design, and scale-up of solid state fungal pretreatment; and 7) the limitations and future perspective of this technology.     

Economic impact of combined torrefaction and pelletization processes on forestry biomass supply

The cost of supplying wood biomass from forestry operations in remote areas has been an obstacle to expansion of forest‐based bioenergy in much of the western United States. Economies of scale in the production of liquid fuels from lignocellulosic biomass feedstocks favor large centralized biorefineries. Increasing transportation efficiency through torrefaction and pelletization at distributed satellite facilities may serve as a means to expand the utilization of forestry residuals in biofuel production. To investigate this potential, a mixed‐integer linear program was developed to optimize the feedstock supply chain design with and without distributed pretreatment. The model uses techno‐economic assessment of scale‐dependent biomass pretreatment processes from existing literature and multimodal biomass transportation cost evaluations derived from a spatially explicit network analysis as input. In addition, the sensitivity of the optimal system configuration was determined for variations of key input parameters including the production scale of pretreatment facilities, road and rail transportation costs, and feedstock procurement costs. Torrefaction and densification were found to reduce transportation costs by $0.84 per GJ and overall delivered costs by $0.24 per GJ, representing 14.5% and 5.2% cost reductions compared to feedstock collection without pretreatment. Significant uncertainties remain in terms of the costs associated with deploying torrefaction equipment at the scales modeled, but the level of potential cost savings suggests further analysis and development of these alternatives.     

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.     

The challenge of enzyme cost in the production of lignocellulosic biofuels

With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.     

Thermoacidophilic proteins for biofuel production

Growing concerns about global climate change and energy dependence have led to an increased effort to reduce carbon emissions. A considerable reduction could be achieved by using biofuels from lignocellulosic biomass instead of fossil fuels. One major bottleneck of biofuel production from lignocellulose is the availability of efficient and inexpensive biocatalysts (i.e. alcohol dehydrogenases, cellulases and esterases) that are active and stable at high temperatures and low pH values. Although heterologous gene expression is used effectively to obtain recombinant proteins derived from mesophiles, the production of thermoacidophilic proteins is often unsuccessful. Some of the reasons for this failure and potential solutions for an increased production of novel extremophilic biocatalysts are discussed here.     

A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.     

New Developments in Chemical Engineering for the Production of Drug Substances

The pharmaceutical industry is moving towards a profitability gap between increasing costs and decreasing prices. Finally, management has understood that mergers and acquisitions, high throughput screening, and biotechnology alone will not save the companies' earnings. Therefore, classical approaches like the optimization of production technologies for drug substances, that might help to increase profitability, are receiving increasing attention. This paper shows how the combination of innovative components will guide the way to very efficient and cost‐effective production. The first component is the design and manufacturing of production facilities. The second component is a process streamlining of the production process. The key technologies discussed here are process control and miniplant technology. For these technologies a brief outlook on future trends is given.     

Methods of downstream processing for the production of biodiesel from microalgae

Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels.     

A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.     

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.     

Hydrodynamic cavitation as a strategy to enhance the efficiency of lignocellulosic biomass pretreatment

Hydrodynamic cavitation (HC) is a process technology with potential for application in different areas including environmental, food processing, and biofuels production. Although HC is an undesirable phenomenon for hydraulic equipment, the net energy released during this process is enough to accelerate certain chemical reactions. The application of cavitation energy to enhance the efficiency of lignocellulosic biomass pretreatment is an interesting strategy proposed for integration in biorefineries for the production of bio-based products. Moreover, the use of an HC-assisted process was demonstrated as an attractive alternative when compared to other conventional pretreatment technologies. This is not only due to high pretreatment efficiency resulting in high enzymatic digestibility of carbohydrate fraction, but also, by its high energy efficiency, simple configuration, and construction of systems, besides the possibility of using on the large scale. This paper gives an overview regarding HC technology and its potential for application on the pretreatment of lignocellulosic biomass. The parameters affecting this process and the perspectives for future developments in this area are also presented and discussed.     

The greenhouse gas emissions performance of cellulosic ethanol supply chains in Europe

Background  

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.     

Do furanic and phenolic compounds of lignocellulosic and algae biomass hydrolyzate inhibit anaerobic mixed cultures? A comprehensive review

Nowadays there is a growing interest on the use of both lignocellulosic and algae biomass to produce biofuels (i.e. biohydrogen, ethanol and methane), as future alternatives to fossil fuels. In this purpose, thermal and thermo-chemical pretreatments have been widely investigated to overcome the natural physico-chemical barriers of such biomass and to enhance biofuel production from lignocellulosic residues and, more recently, marine biomass (i.e. macro and microalgae). However, the pretreatment technologies lead not only to the conversion of carbohydrate polymers (ie cellulose, hemicelluloses, starch, agar) to soluble monomeric sugar (ie glucose, xylose, arabinose, galactose), but also the generation of various by-products (i.e. furfural and 5-HMF). In the case of lignocellulosic residues, part of the lignin can also be degraded in lignin derived by-products, mainly composed of phenolic compounds. Although the negative impact of such by-products on ethanol production has been widely described in literature, studies on their impact on biohydrogen and methane production operated with mixed cultures are still very limited.     

Developments and perspectives of photobioreactors for biofuel production

The production of biofuels from microalgae requires efficient photobioreactors in order to meet the tight constraints of energy efficiency and economic profitability. Current cultivation systems are designed for high-value products rather than for mass production of cheap energy carriers. Future bioreactors will imply innovative solutions in terms of energy efficiency, light and gas transfer or attainable biomass concentration to lower the energy demand and cut down production costs. A new generation of highly developed reactor designs demonstrates the enormous potential of photobioreactors. However, a net energy production with microalgae remains challenging. Therefore, it is essential to review all aspects and production steps for optimization potential. This includes a custom process design according to production organism, desired product and production site. Moreover, the potential of microalgae to synthesize valuable products additionally to the energetic use can be integrated into a production concept as well as waste streams for carbon supply or temperature control.     

Biotreibstoffe aus Entwicklungsländern

Biofuels from developing countries The pressure for reducing greenhouse gas emissions, rising oil prices, but also the lobbying by the agricultural sector and the automotive industry have induced the recent boom on biofuels. Due to limited land availability, competition with food production and high overall environmental impacts, the sustainability market potential for biofuels is assumed to be significantly smaller than 10% of global fuel consumption. Nevertheless, niches for the sustainable production and use of biofuels exist especially in developing countries. It is often more sustainable to use biomass feedstock for local supply of electricity and heat than producing biofuels for export.     

A review of thermal-chemical conversion of lignocellulosic biomass in China

Biomass, a renewable, sustainable and carbon dioxide neutral resource, has received widespread attention in the energy market as an alternative to fossil fuels. Thermal-chemical conversion of biomass to produce biofuels is a promising technology with many commercial applications. This paper reviewed the state-of-the-art research and development of thermal-chemical conversion of biomass in China with a special focus on gasification, pyrolysis, and catalytic transformation technologies. The advantages and disadvantages, potential of future applications, and challenges related to these technologies are discussed. Conclusively, these transformation technologies for the second-generation biofuels with using non-edible lignocellulosic biomass as feedstocks show prosperous perspective for commercial applications in near future.