Microbial removal of nutrients from anaerobic digestate: assessing product-coupled and non-product-coupled approaches.

Although anaerobic digestate contains >90% water, the high nutrient content of digestate makes it economically and technically intractable to treatment by existing wastewater treatment technologies. This study separately assessed the feasibility of nutrient removal from digestate by Rhizopus delemar DSM 905 and a culture of phosphate-accumulating organisms (PAOs). With Rhizopus delemar DSM 905, we investigated concomitant nutrient removal from digestate-supplemented medium and fumaric acid production, as a potentially economical strategy for digestate treatment. Following the cultivation of R. delemar DSM 905 in a fermentation medium containing 25% (v/v) digestate, the concentrations of Al, Cr, Cu, Fe, K, Mg, Mn, Pb, and Zn reduced 40, 12, 74, 96, 12, 26, 23%, ~18, and 28%, respectively. Similarly, the concentrations of total phosphorus, total nitrogen, phosphate (PO4-P), ammonium (NH4-N), nitrate (NO3-N), and sulfur decreased 93, 88, 97, 98, 69, and 13%, respectively. Concomitantly, cultures supplemented with 25 and 15% (v/v) digestate produced comparable titers of fumarate (~11 and ~ 17 g/L, respectively) to the digestate un-supplemented control cultures. With PAOs, we assessed the removal of total phosphorus, total nitrogen, PO4-P, and NH4-N, of which the concentrations reduced 86, 90%, ~99, and 100%, respectively in 60% (v/v) digestate. This study provides additional bases for microbial removal of excess nutrients from anaerobic digestate, with the potential to engender future water recovery from this waste stream that is currently largely recalcitrant to treatment.

Whole-Genome Sequence and Fermentation Characteristics of Enterobacter hormaechei UW0SKVC1: A Promising Candidate for Detoxification of Lignocellulosic Biomass Hydrolysates and Production of Value-Added Chemicals.

Enterobacter hormaechei is part of the Enterobacter cloacae complex (ECC), which is widespread in nature. It is a facultative Gram-negative bacterium of medical and industrial importance. We assessed the metabolic and genetic repertoires of a new Enterobacter isolate. Here, we report the whole-genome sequence of a furfural- and 5-hydroxymethyl furfural (HMF)-tolerant strain of E. hormaechei (UW0SKVC1), which uses glucose, glycerol, xylose, lactose and arabinose as sole carbon sources. This strain exhibits high tolerance to furfural (IC50 = 34.2 mM; ~3.3 g/L) relative to Escherichia coli DH5α (IC50 = 26.0 mM; ~2.5 g/L). Furfural and HMF are predominantly converted to their less-toxic alcohols. E. hormaechei UW0SKVC1 produces 2,3-butanediol, acetoin, and acetol, among other compounds of industrial importance. E. hormaechei UW0SKVC1 produces as high as ~42 g/L 2,3-butanediol on 60 g/L glucose or lactose. The assembled genome consists of a 4,833,490-bp chromosome, with a GC content of 55.35%. Annotation of the assembled genome revealed 4586 coding sequences and 4516 protein-coding genes (average length 937-bp) involved in central metabolism, energy generation, biodegradation of xenobiotic compounds, production of assorted organic compounds, and drug resistance. E. hormaechei UW0SKVC1 shows considerable promise as a biocatalyst and a genetic repository of genes whose protein products may be harnessed for the efficient bioconversion of lignocellulosic biomass, abundant glycerol and lactose-replete whey permeate to value-added chemicals.

Microbial detoxification of lignocellulosic biomass hydrolysates: Biochemical and molecular aspects, challenges, exploits and future perspectives.

Valorization of lignocellulosic biomass (LB) has the potential to secure sustainable energy production without impacting food insecurity, whist relieving over reliance on finite fossil fuels. Agro-derived lignocellulosic residues such as wheat straw, switchgrass, rice bran, and miscanthus have gained relevance as feedstocks for the production of biofuels and chemicals. However, the microorganisms employed in fermentative conversion of carbohydrates to fuels and chemicals are unable to efficiently utilize the sugars derived from LB due to co-production of lignocellulose-derived microbial inhibitory compounds (LDMICs) during LB pretreatment. LDMICs impact microbial growth by inhibition of specific enzymes, cause DNA and cell membrane damage, and elicit cellular redox imbalance. Over the past decade, success has been achieved with the removal of LDMICs prior to fermentation. However, LDMICs removal by chemical processes is often accompanied by sugar losses, which negatively impacts the overall production cost. Hence, in situ removal of LDMICs by fermentative organisms during the fermentation process has garnered considerable attention as the "go-to" approach for economical LDMICs detoxification and bio-chemicals production. In situ removal of LDMICs has been pursued by either engineering more robust biocatalysts or isolating novel microbial strains with the inherent capacity to mineralize or detoxify LDMICs to less toxic compounds. While some success has been made along this line, efficient detoxification and robust production of target bio-chemicals in lignocellulosic hydrolysates (LHs) under largely anaerobic fermentative conditions remains a lingering challenge. Consequently, LB remains an underutilized substrate for bio-chemicals production. In this review, the impact of microbial LH detoxification on overall target molecule production is discussed. Further, the biochemical pathways and mechanisms employed for in situ microbial detoxification of furanic LDMICs [e.g., furfural and 5-hydroxymethylfurfural (HMF)] and phenolic LDMICs (e.g., syringaldehyde, p-coumaric acid, 4-hydroxybenzaldehyde, vanillin, and ferulic acid) are discussed. More importantly, metabolic engineering strategies for the development of LDMIC-tolerant and bio-chemicals overproducing strains and processes are highlighted.

Effects of Clostridium beijerinckii and Medium Modifications on Acetone-Butanol-Ethanol Production From Switchgrass.

The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered C. beijerinckii NCIMB 8052 for the fermentation of LB-derived hydrolysates to acetone-butanol-ethanol (ABE). The engineered microbial strain (C. beijerinckii_SDR) was produced by the integration of an additional copy of a short-chain dehydrogenase/reductase (SDR) gene (Cbei_3904) into the chromosome of C. beijerinckii NCIMB 8052 wildtype, where it is controlled by the constitutive thiolase promoter. The C. beijerinckii_SDR and C. beijerinckii NCIMB 8052 wildtype were used for comparative fermentation of non-detoxified and detoxified hydrothermolysis-pretreated switchgrass hydrolysates (SHs) with and without (NH4)2CO3 supplementation. In the absence of (NH4)2CO3, fermentation of non-detoxified SH with C. beijerinckii_SDR resulted in the production of 3.13- and 2.25-fold greater quantities of butanol (11.21 g/L) and total ABE (20.24 g/L), respectively, than the 3.58 g/L butanol and 8.98 g/L ABE produced by C. beijerinckii_wildtype. When the non-detoxified SH was supplemented with (NH4)2CO3, concentrations were similar for butanol (9.5 compared with 9.2 g/L) and ABE (14.2 compared with 13.5 g/L) produced by C. beijerinckii_SDR and C. beijerinckii_wildtype, respectively. Furthermore, when C. beijerinckii_SDR and C. beijerinckii_wildtype were cultured in detoxified SH medium, C. beijerinckii_SDR produced 1.11- and 1.18-fold greater quantities of butanol and ABE, respectively, than when there was culturing with C. beijerinckii_wildtype. When the combined results of the present study are considered, conclusions are that the microbial strain and medium modifications of the fermentation milieu resulted in greater production of fuels and chemicals from non-detoxified LB hydrolysates.

Complete Genome Sequence of Paenibacillus polymyxa DSM 365, a Soil Bacterium of Agricultural and Industrial Importance.

We report the complete genome sequence of Paenibacillus polymyxa DSM 365. The genome consists of a 5,788,318-bp chromosome, with a GC content of 45.48%. Annotation of the genome revealed a total of 5,246 genes (average length, 943 bp). Gene function analysis indicated the ability to fix nitrogen (N2) and to produce value-added chemicals.

Repurposing anaerobic digestate for economical biomanufacturing and water recovery.

Due to mounting impacts of climate change, particularly increased incidence of drought, hence water scarcity, it has become imperative to develop new technologies for recovering water from nutrient-rich, water-replete effluents other than sewage. Notably, anaerobic digestate could be harnessed for the purpose of water recovery by repurposing digestate-borne minerals as nutrients in fermentative processes. The high concentrations of ammonium, phosphate, sulfate, and metals in anaerobic digestate are veritable microbial nutrients that could be harnessed for bio-production of bulk and specialty chemicals. Tethering nutrient sequestration from anaerobic digestate to bio-product accumulation offers promise for concomitant water recovery, bio-chemical production, and possible phosphate recovery. In this review, we explore the potential of anaerobic digestate as a nutrient source and as a buffering agent in fermentative production of glutamine, glutamate, fumarate, lactate, and succinate. Additionally, we discuss the potential of synthetic biology as a tool for enhancing nutrient removal from anaerobic digestate and for expanding the range of products derivable from digestate-based fermentations. Strategies that harness the nutrients in anaerobic digestate with bio-product accumulation and water recovery could have far-reaching implications on sustainable management of nutrient-rich manure, tannery, and fish processing effluents that also contain high amounts of water. KEY POINTS: • Anaerobic digestate may serve as a source of nutrients in fermentation. • Use of digestate in fermentation would lead to the recovery of valuable water.

Viable strategies for enhancing acetone-butanol-ethanol production from non-detoxified switchgrass hydrolysates.

A process engineering strategy was investigated towards developing a viable scheme for effective conversion of hydrothermolysis pretreated non-detoxified switchgrass hydrolysates (SH) to acetone butanol ethanol (ABE) using a metabolically engineered strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii_AKR. The engineered strain was modified by homologous integration into the chromosome and constitutive expression of Cbei_3974, which encodes an aldo-keto reductase. Intermittent feeding strategy was employed in which fermentation was initiated with 30% of the SH and the remaining 70% SH was added when the optical density (OD600nm) of C. beijerinckii attained 0.5. The ABE (14.9 g/L) produced from non-detoxified SH by the inhibitor-tolerant C. beijerinckii_AKR was comparable to the P2-glucose control medium (14.7 g/L). Using intermittent feeding, wildtype and C. beijerinckii_AKR produced similar amounts of ABE (about 17.5 g/L). This shows that intermittent feeding strategy and C. beijerinckii_AKR enhanced ABE fermentation and eliminated the need for SH detoxification prior to fermentation.

Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii.

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA-Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA-Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA-Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA-Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

Prevalence, distribution and antimicrobial susceptibility pattern of bacterial isolates from a tertiary Hospital in Malawi.

BACKGROUND: Bacterial infections are a significant cause of sickness and death in sub-Saharan Africa. This study aimed at establishing the prevalence, distribution and antimicrobial susceptibility pattern of major bacterial isolates from patients accessing medical care at a tertiary hospital in Malawi. METHODS: We retrospectively reviewed bacteria culture and antimicrobial susceptibility records for 4617 patients from 2002 to 2014 at Mzuzu Central Hospital (MCH). No inclusion and exclusion criteria were followed. Data was analysed using excel (Microsoft office, USA) and GraphPad prism 7 software programs. RESULTS: The most prevalent isolates were S. aureus (34.7%, n = 783), Klebsiella species (17.4%, n = 393) and Proteus species (11.4%, n = 256). Most microorganisms were isolated from adults (88.3%, n = 3889) and pus was the main source (69.3%, n = 1224). S. pneumoniae was predominantly isolated from cerebrospinal fluid (60.3%, n = 44) largely collected from children (88.2%, n = 64). Overall, most bacteria exhibited high resistance to all regularly used antimicrobials excluding ciprofloxacin. CONCLUSIONS: Our report demonstrates an increase in bacterial infection burden in sites other than blood stream and subsequent increase in prevalence of antimicrobial resistance for all major isolates. Creating an epidemiological survey unit at MCH will be essential to help inform better treatment and management options for patients with bacterial infections.

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Chromosomal integration of aldo-keto-reductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii NCIMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory compounds.

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

Metabolic engineering of Clostridium beijerinckii to improve glycerol metabolism and furfural tolerance.

BACKGROUND: Inefficient utilization of glycerol by Clostridium beijerinckii (Cb) is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration, which would in turn, alleviate the toxicity of lignocellulose-derived microbial inhibitory compounds (LDMICs, e.g., furfural), and improve the fermentation of lignocellulosic biomass hydrolysates (LBH) to butanol. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb. RESULTS: By overexpressing two glycerol dehydrogenase (Gldh) genes (dhaD1 and gldA1) from the glycerol hyper-utilizing Clostridium pasteurianum (Cp) as a fused protein in Cb, we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, Cb_dhaD1 + gldA1 achieved a 59% increase in growth, while butanol and acetone-butanol-ethanol (ABE) concentrations and productivities increased 14.0%, 17.3%, and 55.6%, respectively, relative to the control. Co-expression of dhaD1 + gldA1 and gldA1 + dihydroxyacetone kinase (dhaK) resulted in significant payoffs in cell growth and ABE production compared to expression of one Gldh. In the presence of 4-6 g/L furfural, increased glycerol consumption by the dhaD1 + gldA1 strain increased cell growth (> 50%), the rate of furfural detoxification (up to 68%), and ABE production (up to 40%), relative to the plasmid control. Likewise, over-expression of [(dhaD1 + gldA1) dhaK] improved butanol and ABE production by 70% and 50%, respectively, in the presence of 5 and 6 g/L furfural relative to the plasmid control. CONCLUSIONS: Overexpression of Cp gldhs and dhaK in Cb significantly enhanced glycerol utilization, ABE production, and furfural tolerance by Cb. Future research will address the inability of recombinant Cb to metabolize glycerol as a sole substrate.

Fungal wars: The underlying molecular repertoires of combating mycelia.

Non-self contact between fungi elicits strong morphological and biochemical reactions in the mycelia of interacting species. Although these reactions appear to be species- and interaction-specific, some responses such as pigmentation, increased secretion of phenol-oxidases, barrage formation and sealing of the mycelia front are common responses in most interactions. Hence, some species recruit similar molecular machineries in response to non-self. Increasing number of fully sequenced and annotated fungal genomes and advances in genome-wide and global proteome analytical tools now allow researchers to use techniques such as RNA sequencing, micro and macroarray analysis, 2-dimensional protein gel profiling, and differential display of mRNA to probe the underlying molecular mechanisms of combative mycelial interactions. This review provides an overview of the genes and proteins found to be differentially expressed in conflicting fungal mycelia by the use of 'omics' tools. Connections between observed gene and protein repertoires of competing mycelia and the attendant morphological and biochemical changes are presented.

Development of a high-throughput assay for rapid screening of butanologenic strains.

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP+ to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

Investigation of relationship between 2,3-butanediol toxicity and production during growth of Paenibacillus polymyxa.

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded. Cultures of P. polymyxa were then challenged with levo-2,3-BD (20, 40 and 60g/L) at 0h in a glucose medium, and a concentration dependent growth inhibition response to levo-2,3-BD was observed. The growth of P. polymyxa was completely inhibited by 60g/L levo-2,3-BD. Furthermore, P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60g/L at 12, 24 and 36h, respectively) to mimic 2,3-BD accumulation during fermentation. Interestingly, 2,3-BD was reconverted to acetoin when its concentration reached 60g/L, possibly to alleviate 2,3-BD toxicity. Collectively, our findings indicate that 2,3-BD-mediated toxicity is a major metabolic impediment to 2,3-BD overproduction, thus, making it an important metabolic engineering target towards rational design of a 2,3-BD-overproducing strain.

Use of Cupriavidus basilensis-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by Clostridium beijerinckii.

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC(®)BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.

Unorthodox methods for enhancing solvent production in solventogenic Clostridium species.

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest. In addition to attempts at developing LDMIC-tolerant strains via metabolic engineering to enhance utilization of LDS, unconventional approaches that elicit different metabolic perturbations in microorganisms to relieve solvent- and LDMIC-mediated stresses have been explored to increase solvent production from LDS. In this review, the impacts of metabolic perturbations including medium supplementation with glycerol; furfural and 5-hydroxymethyl furfural; allopurinol, an inhibitor of xanthine dehydrogenase; calcium (Ca(2+)) and zinc (Zn(2+)) ions); and artificial electron carriers, methyl viologen and neutral red, on butanol production are discussed. Although these approaches have brought about considerable increases in butanol production, both from LDS and defined glucose-based media, the modes of action for most of these perturbations have yet to be fully characterized. Better understanding of these mechanisms would likely inform development of LDMIC-tolerant, butanol-overproducing strains, as well as possible combinatorial application of these approaches for enhanced butanol production. Hence, delineating the underlying mechanisms of these perturbations deserves further attention.

Butanol production from hydrothermolysis-pretreated switchgrass: Quantification of inhibitors and detoxification of hydrolyzate.

The present study evaluated butanol production from switchgrass based on hydrothermolysis pretreatment. The inhibitors present in the hydrolyzates were measured. Results showed poor butanol production (1g/L) with non-detoxified hydrolyzate. However, adjusting the pH of the non-detoxified hydrolyzate to 6 and adding 4 g/L CaCO3 increased butanol formation to about 6g/L. There was about 1g/L soluble lignin content (SLC), and various levels of furanic and phenolic compounds found in the non-detoxified hydrolyzate. Detoxification of hydrolyzates with activated carbon increased the butanol titer to 11 g/L with a total acetone, butanol and ethanol (ABE) concentration of 17 g/L. These results show the potential of butanol production from hydrothermolysis pretreated switchgrass.