Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences.

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).

Rethinking plastic recycling: A comparison between North America and Europe.

In this article, we identify the problem of plastic proliferation, the consequent expansion of plastic waste in our society, the inadequacies of current attempts to recycle plastic, and the urgency to address this problem in the light of the microplastic threat. It details the problems with current efforts to recycle plastic and the particularly poor recycling rates in North America (NA) when compared to certain countries in the European Union (EU). The obstacles to plastic recycling are overlapping economic, physical and regulatory problems spanning fluctuating resale market prices, residue and polymer contamination and offshore export which often circumvents the entire process. The primary differences between the EU and NA are the costs of end-of-life disposal methods with most EU citizens paying much higher prices for both landfilling and Energy from Waste (incineration) costs compared with NA. At the time of writing, some EU states are either restricted from landfilling mixed plastic waste or the cost is significantly greater than in NA ($80 to 125 USD/t vs $55 USD/t). This makes recycling a favourable option in the EU, and, in turn, has led to more industrial processing and innovation, more recycled product uptake, and the structuring of collection and sorting methods that favour cleaner polymer streams. This is a self-re-enforcing cycle and is evident by EU technologies and industries that have emerged to process "problem plastics", such as mixed plastic film wastes, co-polymer films, thermosets, Polystyrene, (PS) Polyvinyl Chloride (PVC), and others. This is in contrast with NA recycling infrastructure, which has been tailored to shipping low-value mixed plastic waste abroad. Circularity is far from complete in any jurisdiction as export of plastic to developing countries is an opaque, but often used disposal method in the EU as it is in NA. Proposed restrictions on off-shore shipping and regulations requiring minimum recycled plastic content in new products will potentially increase plastic recycling by increasing both supply and demand for recycled product.

In vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella.

Three bacterial species isolated from whole body extracts of the greater wax moth larvae, Galleria mellonella, were evaluated for their ability to utilize low-density polyethylene (LDPE) as a sole carbon source in vitro. These bacteria were identified as Lysinibacillus fusiformis, Bacillus aryabhattai, and Microbacterium oxydans. Their ability to biodegrade LDPE was assessed by growth curves, cell biomass production, polyethylene (PE) weight loss, and the presence of LDPE hydrolysis products in the growth media. Consortia of these bacteria with three other bacteria previously shown to degrade LDPE (Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas putida IRN22) were also tested. Growth curves of the bacteria utilizing LDPE as a sole carbon source revealed a peak in cell density after 24 h. Cell densities declined by 48 h but slowly increased again to different extents, depending on the bacteria. Incubation of LDPE with bacteria isolated from greater wax moth larvae had significant effects on bacterial cell mass production and weight loss of LDPE in PE-containing media. The bacterial consortia were better able to degrade LDPE than were the individual species alone. Gas chromatographic analyses revealed the presence of linear alkanes and other unknown putative LDPE hydrolysis products in some of bacterial culture media.

Microbial degradation of low-density polyethylene and synthesis of polyhydroxyalkanoate polymers.

We have characterized the ability of eight bacterial strains to utilize powdered low-density polyethylene (LDPE) plastic (untreated and without any additives) as a sole carbon source. Cell mass production on LDPE-containing medium after 21 days of incubation varied between 0.083 ± 0.015 g/L cell dry mass (cdm) for Micrococcus luteus IRN20 and 0.39 ± 0.036 g/L for Cupriavidus necator H16. The percent decrease in LDPE mass ranged from 18.9% ± 0.72% for M. luteus IRN20 to 33.7% ± 1.2% for C. necator H16. Linear alkane hydrolysis products from LDPE degradation were detected in the culture media, and the carbon chain lengths of the hydrolysis products detected varied, depending on the species of bacteria. We also determined that C. necator H16 produced short-chain-length polyhydroxyalkanoate biopolymers, while Pseudomonas putida LS46 and Acinetobacter pittii IRN19 produced medium-chain-length biopolymers while growing on polyethylene powder. Cupriavidus necator H16 accumulated poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-V) polymers to 3.18% ± 0.4% of cdm. The monomer composition of the PHB-V was 94.9% ± 0.61% 3-hydroxybutyrate and 5.03% ± 0.56% 3-hydroxyvalerate. This is the first report that provides direct evidence for simultaneous bioconversion of LDPE plastic to biodegradable polyhydroxyalkanoate polymers.

Analysis of carbohydrate-active enzymes in Thermogemmatispora sp. strain T81 reveals carbohydrate degradation ability.

The phylum Chloroflexi is phylogenetically diverse and is a deeply branching lineage of bacteria that express a broad spectrum of physiological and metabolic capabilities. Members of the order Ktedonobacteriales, including the families Ktedonobacteriaceae, Thermosporotrichaceae, and Thermogemmatisporaceae, all have flexible aerobic metabolisms capable of utilizing a wide range of carbohydrates. A number of species within these families are considered cellulolytic and are capable of using cellulose as a sole carbon and energy source. In contrast, Ktedonobacter racemifer, the type strain of the order, does not appear to possess this cellulolytic phenotype. In this study, we confirmed the ability of Thermogemmatispora sp. strain T81 to hydrolyze cellulose, determined the whole-genome sequence of Thermogemmatispora sp. T81, and using comparative bioinformatics analyses, identified genes encoding putative carbohydrate-active enzymes (CAZymes) in the Thermogemmatispora sp. T81, Thermogemmatispora onikobensis, and Ktedonobacter racemifer genomes. Analyses of the Thermogemmatispora sp. T81 genome identified 64 CAZyme gene sequences belonging to 57 glycoside hydrolase families. The genome of Thermogemmatispora sp. T81 encodes 19 genes for putative extracellular CAZymes, similar to the number of putative extracellular CAZymes identified in T. onikobensis (17) and K. racemifer (17), despite K. racemifer not possessing a cellulolytic phenotype. These results suggest that these members of the order Ktedonobacteriales may use a broader range of carbohydrate polymers than currently described.

Lignin biosynthesis in wheat (Triticum aestivum L.): its response to waterlogging and association with hormonal levels.

BACKGROUND: Lignin is an important structural component of plant cell wall that confers mechanical strength and tolerance against biotic and abiotic stressors; however it affects the use of biomass such as wheat straw for some industrial applications such as biofuel production. Genetic alteration of lignin quantity and quality has been considered as a viable option to overcome this problem. However, the molecular mechanisms underlying lignin formation in wheat biomass has not been studied. Combining molecular and biochemical approaches, the present study investigated the transcriptional regulation of lignin biosynthesis in two wheat cultivars with varying lodging characteristics and also in response to waterlogging. It also examined the association of lignin level in tissues with that of plant hormones implicated in the control of lignin biosynthesis. RESULTS: Analysis of lignin biosynthesis in the two wheat cultivars revealed a close association of lodging resistance with internode lignin content and expression of 4-coumarate:CoA ligase1 (4CL1), p-coumarate 3-hydroxylase1 (C3H1), cinnamoyl-CoA reductase2 (CCR2), ferulate 5-hydroxylase2 (F5H2) and caffeic acid O-methyltransferase2 (COMT2), which are among the genes highly expressed in wheat tissues, implying the importance of these genes in mediating lignin deposition in wheat stem. Waterlogging of wheat plants reduced internode lignin content, and this effect is accompanied by transcriptional repression of three of the genes characterized as highly expressed in wheat internode including phenylalanine ammonia-lyase6 (PAL6), CCR2 and F5H2, and decreased activity of PAL. Expression of the other genes was, however, induced by waterlogging, suggesting their role in the synthesis of other phenylpropanoid-derived molecules with roles in stress responses. Moreover, difference in internode lignin content between cultivars or change in its level due to waterlogging is associated with the level of cytokinin. CONCLUSION: Lodging resistance, tolerance against biotic and abiotic stresses and feedstock quality of wheat biomass are closely associated with its lignin content. Therefore, the findings of this study provide important insights into the molecular mechanisms underlying lignin formation in wheat, an important step towards the development of molecular tools that can facilitate the breeding of wheat cultivars for optimized lignin content and enhanced feedstock quality without affecting other lignin-related agronomic benefits.

Transcriptomic and proteomic analyses of core metabolism in Clostridium termitidis CT1112 during growth on α-cellulose, xylan, cellobiose and xylose.

BACKGROUND: Clostridium termitidis CT1112 is an anaerobic, Gram-positive, mesophilic, spore-forming, cellulolytic bacterium, originally isolated from the gut of a wood feeding termite Nasusitermes lujae. It has the ability to hydrolyze both cellulose and hemicellulose, and ferment the degradation products to acetate, formate, ethanol, lactate, H2, and CO2. It is therefore ges in gene and gene product expression during growth of C. termitidis on cellobiose, xylose, xylan, and α-cellulose. RESULTS: Correlation of transcriptome and proteome data with growth and fermentation profiles identified putative carbon-catabolism pathways in C. termitidis. The majority of the proteins associated with central metabolism were detected in high abundance. While major differences were not observed in gene and gene-product expression for enzymes associated with metabolic pathways under the different substrate conditions, xylulokinase and xylose isomerase of the pentose phosphate pathway were found to be highly up-regulated on five carbon sugars compared to hexoses. In addition, genes and gene-products associated with a variety of cellulosome and non-cellulosome associated CAZymes were found to be differentially expressed. Specifically, genes for cellulosomal enzymes and components were highly expressed on α-cellulose, while xylanases and glucosidases were up-regulated on 5 carbon sugars with respect to cellobiose. Chitinase and cellobiophosphorylases were the predominant CAZymes expressed on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also demonstrated. CONCLUSION: There are little changes in core-metabolic pathways under the different carbon sources compared. The most significant differences were found to be associated with the CAZymes, as well as specific up regulation of some key components of the pentose phosphate pathway in the presence of xylose and xylan. This study has enhanced our understanding of the physiology and metabolism of C. termitidis, and provides a foundation for future studies on metabolic engineering to optimize biofuel production from natural biomass.

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum.

Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

Optimization of influential nutrients during direct cellulose fermentation into hydrogen by Clostridium thermocellum.

Combinatorial effects of influential growth nutrients were investigated in order to enhance hydrogen (H2) production during direct conversion of cellulose by Clostridium thermocellum DSM 1237. A central composite face-centered design and response surface methodology (RSM) were applied to optimize concentrations of cellulose, yeast extract (YE), and magnesium chloride (Mg) in culture. The overall optimum composition generated by the desirability function resulted in 57.28 mmol H2/L-culture with 1.30 mol H2/mol glucose and 7.48 mmol/(g·cell·h) when cultures contained 25 g/L cellulose, 2 g/L YE, and 1.75 g/L Mg. Compared with the unaltered medium, the optimized medium produced approximately 3.2-fold more H2 within the same time-frame with 50% higher specific productivity, which are also better than previously reported values from similar studies. Nutrient composition that diverted carbon and electron flux away from H2 promoting ethanol production was also determined. This study represents the first investigation dealing with multifactor optimization with RSM for H2 production during direct cellulose fermentation.

Thermoanaerobacter thermohydrosulfuricus WC1 shows protein complement stability during fermentation of key lignocellulose-derived substrates.

Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using "omic"-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species "omic" comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.

Characterization of enriched aerotolerant cellulose-degrading communities for biofuels production using differing selection pressures and inoculum sources.

Ethanol production from direct cellulose fermentation has mainly been described as a strictly anaerobic process. The use of air-tolerant organisms or consortia for this process would reduce the need for prereduction of the medium and also permit continuous feed of aerobic feedstock. To this end, moderately thermophilic (60 °C) consortia of fermentative, cellulolytic bacteria were enriched from 3 distinct environments (manure, marsh, and rotten wood) from a farm in southeast Saskatchewan, Canada. Community phenotypic and metabolic profiles were characterized. Selection methods included direct plating under an aerobic atmosphere and repeated passaging; the methods were designed to select for robust, stable aerotolerant cellulose-degrading communities. Several of the isolated communities exhibited an increase in total cellulose degradation and total ethanol yield when compared with a monoculture of Clostridium thermocellum DSMZ 1237. Owing to stringent selection conditions, low diversity enrichments were found, and many appeared to be binary cultures via density gradient gel electrophoresis analysis. On the basis of 16S rRNA gene sequencing, aerobic conditions selected for a mix of organisms highly related to C. thermocellum and Geobacillus species, while anaerobic conditions led to the development of consortia containing strains related to C. thermocellum with strains from either the genus Geobacillus or the genus Thermoanaerobacter. The presence of a Geobacillus-like species appeared to be a prerequisite for aerotolerance of the cellulolytic enrichments, a highly desired phenotype in lignocellulosic consolidated bioprocessing.

Enzyme Systems of Anaerobes for Biomass Conversion.

Biofuels from abundantly available cellulosic biomass are an attractive alternative to current petroleum-based fuels (fossil fuels). Although several strategies exist for commercial production of biofuels, conversion of biomass to biofuels via consolidated bioprocessing offers the potential to reduce production costs and increase processing efficiencies. In consolidated bioprocessing (CBP), enzyme production, cellulose hydrolysis, and fermentation are all carried out in a single-step by microorganisms that efficiently employ a multitude of intricate enzymes which act synergistically to breakdown cellulose and its associated cell wall components. Various strategies employed by anaerobic cellulolytic bacteria for biomass hydrolysis are described in this chapter. In addition, the regulation of CAZymes, the role of "omics" technologies in assessing lignocellulolytic ability, and current strategies for improving biomass hydrolysis for optimum biofuel production are highlighted.

Comparative Genomics of Core Metabolism Genes of Cellulolytic and Non-cellulolytic Clostridium Species.

Microbial production of fuels such as ethanol, butanol, hydrogen (H2), and methane (CH4) from waste biomass has the potential to provide sustainable energy systems that can displace fossil fuel consumption. Screening for microbial diversity and genome sequencing of a wide-range of microorganisms can identify organisms with natural abilities to synthesize these alternative fuels and/or other biotechnological applications. Clostridium species are the most widely studied strict anaerobes capable of fermentative synthesis of ethanol, butanol, or hydrogen directly from waste biomass. Clostridium termitidis CT1112 is a mesophilic, cellulolytic species capable of direct cellulose fermentation to ethanol and organic acids, with concomitant synthesis of H2 and CO2. On the basis of 16S ribosomal RNA (rRNA) and chaperonin 60 (cpn60) gene sequence data, phylogenetic analyses revealed a close relationship between C. termitidis and C. cellobioparum. Comparative bioinformatic analyses of the C. termitidis genome with 18 cellulolytic and 10 non-cellulolytic Clostridium species confirmed this relationship, and further revealed that the majority of core metabolic pathway genes in C. termitidis and C. cellobioparum share more than 90% amino acid sequence identity. The gene loci and corresponding amino acid sequences of the encoded enzymes for each pathway were correlated by percentage identity, higher score (better alignment), and lowest e-value (most significant "hit"). In addition, the function of each enzyme was proposed by conserved domain analysis. In this chapter we discuss the comparative analysis of metabolic pathways involved in synthesis of various useful products by cellulolytic and non-cellulolytic biofuel and solvent producing Clostridium species. This study has generated valuable information concerning the core metabolism genes and pathways of C. termitidis CT1112, which is helpful in developing metabolic engineering strategies to enhance its natural capacity for better industrial applications.

Quantitative proteomic analysis of the cellulolytic system of Clostridium termitidis CT1112 reveals distinct protein expression profiles upon growth on α-cellulose and cellobiose.

Clostridium termitidis CT1112 is an anaerobic, mesophilic, cellulolytic bacterium with potential applications in consolidated bioprocessing of lignocellulosic biomass. To understand how C. termitidis degrades lignocellulose, iTRAQ-based 2D HPLC-MS/MS proteomics was used to measure protein expression in cell lysates and extracellular (secretome) fractions of C. termitidis grown on α-cellulose and cellobiose at both exponential and stationary growth phases. Exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), hemicellulases including xylanases (GH8, GH10, GH11, GH30) and mannanase (GH26) as well as extracellular adhesion proteins and cellulosome associated proteins, exhibited higher expression on cellulose-grown cells. The expression of these proteins increased with a decrease in growth rate. Non-cellulosomal proteins however did not change significantly between substrate conditions, although there were a few exceptions. Collectively, these would contribute to hydrolysis of lignocellulosic material for uptake through ABC sugar transport proteins. On cellobiose, chitinases (GH18) were expressed abundantly. Although a large number of proteins were shared between the fractions analyzed, some proteins were detected exclusively in the cellular fraction, while others were detected in the secretome. This study reports for the first time on the cellulolytic machinery employed by C. termitidis to hydrolyze cellulosic substrate and provides an understanding of how this microbe deconstructs biomass. BIOLOGICAL SIGNIFICANCE: The genome of C. termitidis CT1112 contains genes for a wide variety of carbohydrate active enzymes. Based on bioinformatics analyses, many of these genes appear to encode cellulosome-associated proteins, while others may be secreted extracellularly. To understand how C. termitidis degrades and depolymerizes cellulosic substrates, cells were grown on simple and complex carbohydrates, and quantitative 4-plex iTRAQ-based 2D HPLC-MS/MS proteomics was applied to measure protein expression levels in biological replicates of both cell lysates and extracellular protein (secretome) fractions, at exponential and stationary phases of growth. The resulting data have provided insight into the range of substrates that may be hydrolyzed by C. termitidis, and may be useful in determining potential industrial applications of C. termitidis in biomass to bioenergy production via consolidated bioprocessing.

Recent patents on genetic modification of plants and microbes for biomass conversion to biofuels.

Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort. Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials, but pre-treatment of the biomass to release sugars for microbial conversion is a significant barrier to commercial success of lignocellulosic biofuel production. Strategies to reduce the energy and cost inputs required for biomass pre-treatment include genetic modification of plant materials to reduce lignin content. Significant efforts are also underway to create recombinant microorganisms capable of converting sugars derived from lignocellulosic biomass to a variety of biofuels. An alternative strategy to reduce the costs of cellulosic biofuel production is the use of cellulolytic microorganisms capable of direct microbial conversion of ligno-cellulosic biomass to fuels. This paper reviews recent patents on genetic modification of plants and microbes for biomass conversion to biofuels.

Genomic evaluation of Thermoanaerobacter spp. for the construction of designer co-cultures to improve lignocellulosic biofuel production.

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.

Biomass pretreatment: fundamentals toward application.

Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort. Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials, but the biomass must be subjected to pretreatment processes to liberate the sugars needed for fermentation. Production of value-added co-products along-side biofuels through integrated biorefinery processes creates the need for selectivity during pretreatment. This paper presents a survey of biomass pretreatment technologies with emphasis on concepts, mechanism of action and practicability. The advantages and disadvantages, and the potential for industrial applications of different pretreatment technologies are the highlights of this paper.