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.
Bioethanol; Biological pre-treatment; Delignification; Lignocellulose
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.
Consortia; Consolidated bioprocessing; Cellulosic ethanol; Symbiosis; Oxygen transport
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.
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.
cellulosic ethanol; consolidated bioprocessing; integrating bio-based industries; sustainable biofuels in Africa
The growing need to address current energy and environmental problems has sparked an interest in developing improved biological methods to produce liquid fuels from renewable sources. While microbial ethanol production is well established, higher-chain alcohols possess chemical properties that are more similar to gasoline. Unfortunately, these alcohols (except 1-butanol) are not produced efficiently in natural microorganisms, and thus economical production in industrial volumes remains a challenge. Synthetic biology, however, offers additional tools to engineer synthetic pathways in user-friendly hosts to help increase titers and productivity of these advanced biofuels. This review concentrates on recent developments in synthetic biology to produce higher-chain alcohols as viable renewable replacements for traditional fuel.
biofuel; butanol; higher-chain alcohol; isobutanol; metabolic engineering; synthetic biology
Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.
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.
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.
Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.
In this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.
Overall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.
Lignin is one of the three major components in plant cell walls, and it can be isolated (dissolved) from the cell wall in pretreatment or chemical pulping. However, there is a lack of high-value applications for lignin, and the commonest proposal for lignin is power and steam generation through combustion. Organosolv ethanol process is one of the effective pretreatment methods for woody biomass for cellulosic ethanol production, and kraft process is a dominant chemical pulping method in paper industry. In the present research, the lignins from organosolv pretreatment and kraft pulping were evaluated to replace polyol for producing rigid polyurethane foams (RPFs).
Petroleum-based polyol was replaced with hardwood ethanol organosolv lignin (HEL) or hardwood kraft lignin (HKL) from 25% to 70% (molar percentage) in preparing rigid polyurethane foam. The prepared foams contained 12-36% (w/w) HEL or 9-28% (w/w) HKL. The density, compressive strength, and cellular structure of the prepared foams were investigated and compared. Chain extenders were used to improve the properties of the RPFs.
It was found that lignin was chemically crosslinked not just physically trapped in the rigid polyurethane foams. The lignin-containing foams had comparable structure and strength up to 25-30% (w/w) HEL or 19-23% (w/w) HKL addition. The results indicated that HEL performed much better in RPFs and could replace more polyol at the same strength than HKL because the former had a better miscibility with the polyol than the latter. Chain extender such as butanediol could improve the strength of lignin-containing RPFs.
Kraft lignin; Lignin utilization; Organosolv lignin; Polyurethane; Rigid foam
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.
Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of 'intelligent' metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.
Fermentation; Bioethanol; Lignocellulosic Inhibitors; Lignocellulolytic materials; Stress Response; Microbial Physiology; Phenolics.
Solventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. C. beijerinckii is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating C. beijerinckii metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications.
We present the first genome-scale metabolic model (iCM925) for C. beijerinckii, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate iCM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (P = 3.52 × 10-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed.
Microbial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production.
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.
The microbial production of ethanol from lignocellulosic biomass is a multi-component process that involves biomass hydrolysis, carbohydrate transport and utilization, and finally, the production of ethanol. Strains of the genus Thermoanaerobacter have been studied for decades due to their innate abilities to produce comparatively high ethanol yields from hemicellulose constituent sugars. However, their inability to hydrolyze cellulose, limits their usefulness in lignocellulosic biofuel production. As such, co-culturing Thermoanaerobacter spp. with cellulolytic organisms is a plausible approach to improving lignocellulose conversion efficiencies and yields of biofuels. To evaluate native lignocellulosic ethanol production capacities relative to competing fermentative end-products, comparative genomic analysis of 11 sequenced Thermoanaerobacter strains, including a de novo genome, Thermoanaerobacter thermohydrosulfuricus WC1, was conducted. Analysis was specifically focused on the genomic potential for each strain to address all aspects of ethanol production mentioned through a consolidated bioprocessing approach. Whole genome functional annotation analysis identified three distinct clades within the genus. The genomes of Clade 1 strains encode the fewest extracellular carbohydrate active enzymes and also show the least diversity in terms of lignocellulose relevant carbohydrate utilization pathways. However, these same strains reportedly are capable of directing a higher proportion of their total carbon flux towards ethanol, rather than non-biofuel end-products, than other Thermoanaerobacter strains. Strains in Clade 2 show the greatest diversity in terms of lignocellulose hydrolysis and utilization, but proportionately produce more non-ethanol end-products than Clade 1 strains. Strains in Clade 3, in which T. thermohydrosulfuricus WC1 is included, show mid-range potential for lignocellulose hydrolysis and utilization, but also exhibit extensive divergence from both Clade 1 and Clade 2 strains in terms of cellular energetics. The potential implications regarding strain selection and suitability for industrial ethanol production through a consolidated bioprocessing co-culturing approach are examined throughout the manuscript.
Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.
Increasing public awareness of environmental pollution influences the search and development of technologies that help in clean up of organic and inorganic contaminants such as hydrocarbons and metals. An alternative and eco-friendly method of remediation technology of environments contaminated with these pollutants is the use of biosurfactants and biosurfactant-producing microorganisms. The diversity of biosurfactants makes them an attractive group of compounds for potential use in a wide variety of industrial and biotechnological applications. The purpose of this review is to provide a comprehensive overview of advances in the applications of biosurfactants and biosurfactant-producing microorganisms in hydrocarbon and metal remediation technologies.
biosurfactants; hydrocarbons; metals; remediation technologies
Marine microorganisms are rich source for natural products which play important roles in pharmaceutical industry. Over the past decade, genome-based studies of marine microorganisms have unveiled the tremendous diversity of the producers of natural products and also contributed to the efficiency of harness the strain diversity and chemical diversity, as well as the genetic diversity of marine microorganisms for the rapid discovery and generation of new natural products. In the meantime, genomic information retrieved from marine symbiotic microorganisms can also be employed for the discovery of new medical molecules from yet-unculturable microorganisms. In this paper, the recent progress in the genomic research of marine microorganisms is reviewed; new tools of genome mining as well as the advance in the activation of orphan pathways and metagenomic studies are summarized. Genome-based research of marine microorganisms will maximize the biodiscovery process and solve the problems of supply and sustainability of drug molecules for medical treatments.
An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.
In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).
The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.
Solar energy is potentially the largest source of renewable energy at our disposal, but significant advances are required to make photovoltaic technologies economically viable and, from a life-cycle perspective, environmentally friendly, and consequently scalable. Cellulose nanomaterials are emerging high-value nanoparticles extracted from plants that are abundant, renewable, and sustainable. Here, we report on the first demonstration of efficient polymer solar cells fabricated on optically transparent cellulose nanocrystal (CNC) substrates. The solar cells fabricated on the CNC substrates display good rectification in the dark and reach a power conversion efficiency of 2.7%. In addition, we demonstrate that these solar cells can be easily separated and recycled into their major components using low-energy processes at room temperature, opening the door for a truly recyclable solar cell technology. Efficient and easily recyclable organic solar cells on CNC substrates are expected to be an attractive technology for sustainable, scalable, and environmentally-friendly energy production.
Fermentation production of biofuel ethanol consumes agricultural crops, which will compete directly with the food supply. As an alternative, photosynthetic cyanobacteria have been proposed as microbial factories to produce ethanol directly from solar energy and CO2. However, the ethanol productivity from photoautotrophic cyanobacteria is still very low, mostly due to the low tolerance of cyanobacterial systems to ethanol stress.
To build a foundation necessary to engineer robust ethanol-producing cyanobacterial hosts, in this study we applied a quantitative transcriptomics approach with a next-generation sequencing technology, combined with quantitative reverse-transcript PCR (RT-PCR) analysis, to reveal the global metabolic responses to ethanol in model cyanobacterial Synechocystis sp. PCC 6803. The results showed that ethanol exposure induced genes involved in common stress responses, transporting and cell envelope modification. In addition, the cells can also utilize enhanced polyhydroxyalkanoates (PHA) accumulation and glyoxalase detoxication pathway as means against ethanol stress. The up-regulation of photosynthesis by ethanol was also further confirmed at transcriptional level. Finally, we used gene knockout strains to validate the potential target genes related to ethanol tolerance.
RNA-Seq based global transcriptomic analysis provided a comprehensive view of cellular response to ethanol exposure. The analysis provided a list of gene targets for engineering ethanol tolerance in cyanobacterium Synechocystis.
Ethanol; Tolerance; Transcriptomics; Synechocystis
Lignocellulosic biomass, such as corn stover, is a potential raw material for ethanol production. One step in the process of producing ethanol from lignocellulose is enzymatic hydrolysis, which produces fermentable sugars from carbohydrates present in the corn stover in the form of cellulose and hemicellulose. A pretreatment step is crucial to achieve efficient conversion of lignocellulosic biomass to soluble sugars, and later ethanol. This study has investigated steam pretreatment of corn stover, with and without sulphuric acid as catalyst, and examined the effect of residence time (5–10 min) and temperature (190–210°C) on glucose and xylose recovery. The pretreatment conditions with and without dilute acid that gave the highest glucose yield were then used in subsequent experiments. Materials pretreated at the optimal conditions were subjected to simultaneous saccharification and fermentation (SSF) to produce ethanol, and remaining organic compounds were used to produce biogas by anaerobic digestion (AD).
The highest glucose yield achieved was 86%, obtained after pretreatment at 210°C for 10 minutes in the absence of catalyst, followed by enzymatic hydrolysis. The highest yield using sulphuric acid, 78%, was achieved using pretreatment at 200°C for 10 minutes. These two pretreatment conditions were investigated using two different process configurations. The highest ethanol and methane yields were obtained from the material pretreated in the presence of sulphuric acid. The slurry in this case was split into a solid fraction and a liquid fraction, where the solid fraction was used to produce ethanol and the liquid fraction to produce biogas. The total energy recovery in this case was 86% of the enthalpy of combustion energy in corn stover.
The highest yield, comprising ethanol, methane and solids, was achieved using pretreatment in the presence of sulphuric acid followed by a process configuration in which the slurry from the pretreatment was divided into a solid fraction and a liquid fraction. The solid fraction was subjected to SSF, while the liquid fraction, together with the filtered residual from SSF, was used in AD. Using sulphuric acid in AD did not inhibit the reaction, which may be due to the low concentration of sulphuric acid used. In contrast, a pretreatment step without sulphuric acid resulted not only in higher concentrations of inhibitors, which affected the ethanol yield, but also in lower methane production.
Corn stover; Steam pretreatment; Ethanol; Methane; Sulphuric acid
Clostridium thermocellum produces ethanol, acetate, H2, and CO2 as major fermentation products from cellulose and cellobiose. The performance of three strains of this microorganism was studied to assess the potential use in producing ethanol directly from cellulosic fiber. Depending on the bacterial strain, an ethanol/acetate product ratio from 1 to as high as 3 was observed in unstirred cultures. Vigorous stirring during growth resulted in a threefold decrease in the ethanol/acetate ratio. The H2 content in the unstirred culture broth was three times greater than that in the stirred one. Addition of exogenous H2 to the gas phase during growth increased the ethanol/acetate ratio much more in the stirred than in the unstirred fermentations. The addition of sufficient H2 to the gas phase almost relieved the effect of stirring, and the ethanol/acetate ratio approached that in the unstirred condition. Addition of tritium to the gas phase of the culture resulted in the formation of tritiated water (3H2O), which indicates that C. thermocellum possesses hydrogenase(s) that catalyzes the reverse reaction. The rate of 3H2O formation was about three times higher in the stirred culture than in the unstirred culture. These results demonstrate that the H2 concentration in the broth plays an important role in the product formation. The H2 supersaturation present in the unstirred cultures is responsible for the observed effect of stirring. A hydrogen feedback control mechanism regulating the relative concentrations of reduced and oxidized electron carriers is proposed to account for the effect of hydrogen on the metabolite distribution.
Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold.
We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance.
Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.
Switchgrass; Bioenergy; Biofuel; Feedstock; Cellulosic ethanol; PvMYB4; Transcription factor; Cell wall; Recalcitrance; Lignin; Hemicellulose; Pectin
Adhesion and accumulation of organic molecules represent an ecologically and economically massive problem. Adhesion of organic molecules is followed by microorganisms, unicellular organisms and plants together with their secreted soluble and structure-associated byproducts, which damage unprotected surfaces of submerged marine structures, including ship hulls and heat exchangers of power plants. This is termed biofouling. The search for less toxic anti-biofilm strategies has intensified since the ban of efficient and cost-effective anti-fouling paints, enriched with the organotin compound tributyltin, not least because of our finding of the ubiquitous toxic/pro-apoptotic effects displayed by this compound . Our proposed bio-inspired approach for controlling, suppressing and interfluencing the dynamic biofouling complex uses copper as one component in an alternative anti-fouling system. In order to avoid and overcome the potential resistance against copper acquired by microorganisms we are using the biopolymer polyphosphate (polyP) as a further component. Prior to being functionally active, polyP has to be hydrolyzed to ortho-phosphate which in turn can bind to copper and export the toxic compound out of the cell. It is shown here that inhibition of the hydrolysis of polyP by the bisphosphonate DMDP strongly increases the toxic effect of copper towards the biofilm-producing Streptococcus mutans in a synergistic manner. This bisphosphonate not only increases the copper-caused inhibition of cell growth but also of biofilm production by the bacteria. The defensin-related ASABF, a marine toxin produced by the sponge Suberites domuncula, caused only an additive inhibitory effect in combination with copper. We conclude that the new strategy, described here, has a superior anti-biofilm potential and can be considered as a novel principle for developing bio-inspired antifouling compounds, or cocktails of different compounds, in the future.
biofilm; marine coatings; antifouling strategies; synergism; polyphosphate; copper; bisphosphonate; bioinspired approach