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The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

Since 2011, unprecedented beaching events of Sargassum seaweed have caused major environmental, health and economic problems in the Caribbean, Gulf of Mexico, northern Brazil and the western coast of Africa. Not only are Sargassum influxes threatening already fragile and often endangered coastal ecosystems, such as coral reefs, mangroves and seagrass beds, they also disrupt the livelihoods of communities, especially those associated with the tourism and fishing sectors. The aim of this study is to develop a plan to turn these “brown tides” into opportunities for sustainable, scalable and efficient harvesting and valorisation approaches that will deliver environmental and socioeconomic benefits to the Dutch Caribbean and other end-users in the region. In this report we have reviewed the state-of-the-art of research on recent Sargassum blooms and influxes in the Caribbean with respect to biology, ecology, origin, distribution, socio-ecological impact and management options, in addition to existing valorisation chains and uses of Sargassum biomass, and environmental and socio-economic impacts of Sargassum valorisation. We conclude that value chains based on valorisation of nearshore Sargassum biomass into biofuel and agricultural products (i.e. fertilizer, animal feed supplement) seem the most promising for the Dutch Caribbean islands, since they will contribute to sustainable energy and food security, while reducing environmental impact of the energy and agricultural sector. Some of the identified management and valorisation strategies could also be applied to other areas that are affected by massive Sargassum influxes, such as the Gulf of Mexico. During 2020 pelagic Sargassum samples have been collected in different locations in the Caribbean region (Bonaire, Mexico, St Maarten) and Florida. These samples have been analysed for major components (including sugars and ash) and for content in iodine and heavy metals. The values obtained in these analysis were compared to values reported in the literature. The results of these analysis are of importance for the definition of possible applications of the Sargassum biomass or its products as component in the food and feed value chains Based on our literature review, a number of knowledge gaps have been identified that are related to the availability of pelagic Sargassum biomass, the environmental impacts of the harvesting, technological challenges, effects of Sargassum leachates to the environment (in case of agricultural uses) and on socio-economic impacts of the current and predicted actions (Chapter 7). These knowledge gaps need to be addressed before (commercial) harvesting and valorisation actions concerning pelagic Sargassum are taken. Therefore, we defined an implementation plan (Section 7.5 and Fig. 1), which addresses these knowledge gaps to set the first steps in the short term towards efficient and sustainable management and valorisation of pelagic Sargassum in the Dutch Caribbean. In the potential value chains defined, prevention of landings of Sargassum on the coasts is preferred to harvesting onshore. This is due to the negative ecological impact of harvesting onshore and the lower quality of beached Sargassum, that decays very rapidly.

International audience; Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6 kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general., Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose–l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose. IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

The growing world population demands an increase in animal protein production. Seaweed may be a valuable source of protein for animal feed. However, a biorefinery approach aimed at cascading valorisation of both protein and non-protein seaweed constituents is required to realise an economically feasible value chain. In this study, such a biorefinery approach is presented for the green seaweed Ulva lactuca containing 225 g protein (N × 4.6) kg−1 dry matter (DM). The sugars in the biomass were solubilised by hot water treatment followed by enzymatic hydrolysis and centrifugation resulting in a sugar-rich hydrolysate (38.8 g L−1 sugars) containing glucose, rhamnose and xylose, and a protein-enriched (343 g kg−1 in DM) extracted fraction. This extracted fraction was characterised for use in animal feed, as compared to U. lactuca biomass. Based on the content of essential amino acids and the in vitro N (85 %) and organic matter (90 %) digestibility, the extracted fraction seems a promising protein source in diets for monogastric animals with improved characteristics as compared to the intact U. lactuca. The gas production test indicated a moderate rumen fermentation of U. lactuca and the extracted fraction, about similar to that of alfalfa. Reduction of the high content of minerals and trace elements may be required to allow a high inclusion level of U. lactuca products in animal diets. The hydrolysate was used successfully for the production of acetone, butanol, ethanol and 1,2-propanediol by clostridial fermentation, and the rhamnose fermentation pattern was studied. Electronic supplementary material The online version of this article (doi:10.1007/s10811-016-0842-3) contains supplementary material, which is available to authorized users.

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

An exploratory Life Cycle Assessment was carried out to provide insight into the environmental impacts of using the green seaweed Ulva spp. as a feedstock for production of bioplastic. The study focused on the production of lactic acid as a precursor of polylactic acid. The studied production process consists of (1) the cultivation of Ulva spp. in Integrated Multitrophic Aquaculture, (2) the processing of the biomass for solubilisation of sugars, (3) the fermentation of the sugars to lactic acid and (4) isolation of lactic acid from the fermentation broth. The study identifies environmental hotspots and compares the experimental seaweed production chain with conventional feedstock. The main hotspot identified is electricity consumption during seaweed cultivation. This impact of electricity consumption can be lowered by reducing energy use and sourcing renewable energy, and by improving the material efficiency in the product chain. To improve understanding of the environmental impacts of the production process, future studies can broaden the system boundaries and broaden the scope of sustainability issues included in the environmental assessment.

An exploratory Life Cycle Assessment (LCA) was carried out to provide insight into the environmental impacts of using the green seaweed Ulva spp. as a feedstock, for production of bioplastic. The study focused on the production of lactic acid as a precursor of polylactic acid. The study was on the production process: (1) The cultivation of Ulva spp., in an Integrated Multitrophic Aquaculture system, (2) the processing of the biomass for solubilization of sugars, (3) the fermentation of the sugars to lactic acid, (4) the isolation of lactic acid from fermentation broth. The study identified environmental hotspots and compared an experimental seaweed production chain with conventional feedstocks. The main hotspot is derived from electricity consumption during seaweed cultivation. The impact of electricity consumption can be lowered by reducing energy use and sourcing renewable energy, and by improving the material efficiency in the product chain. To improve understanding of the process of production&rsquo, s environmental impacts, future studies should broaden the system boundaries and scope of sustainability issues included in the environmental assessment.

Background There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (

Background There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (

Green seaweed Ulva lactuca harvested from the North Sea near Zeeland (The Netherlands) was characterized as feedstock for acetone, ethanol and ethanol fermentation. Solubilization of over 90% of sugars was achieved by hot-water treatment followed by hydrolysis using commercial cellulases. A hydrolysate was used for the production of acetone, butanol and ethanol (ABE) by Clostridium acetobutylicum and Clostridium beijerinckii. Hydrolysate-based media were fermentable without nutrient supplementation. C. beijerinckii utilized all sugars in the hydrolysate and produced ABE at high yields (0.35 g ABE/g sugar consumed), while C. acetobutylicum produced mostly organic acids (acetic and butyric acids). These results demonstrate the great potential of U. lactuca as feedstock for fermentation. Interestingly, in control cultures of C. beijerinckii on rhamnose and glucose, 1,2 propanediol was the main fermentation product (9.7 g/L).

In this study, the removal of IBE from aqueous solutions by gas stripping has been characterized. The effect of one or more components in the solution on the kinetics of the separation has been studied, both at 37 °C and at 70 °C. Gas stripping has been applied to batch, repeated batch and continuous cultures of Clostridium beijerinckii grown on a glucose/xylose mixed sugar substrate mimicking lignocellulosic hydrolysates, with the aim of finding optimal conditions for a stable IBE-producing culture with high productivity. An innovative repeated-batch process has been demonstrated in which the gas-stripping is performed at 70 °C, resulting in a prolonged stable IBE culture.

Presentation of carbohydrate analysis of seaweed in the biorefinery to chemicals and fuel context, presented at the International Seaweed Symposium 2016, Copenhagen, Denmark, 19-24 June 2016.

Biomass fermentation to Iso-propanol, Butanol and Ethanol (IBE) is particularly important as IBE is a common building block in the development of biorefineries and IBE-producing bacteria are robust industrial organisms, capable to utilize the sugars of the lignocellulosic biomass. Research is focused on increasing fermentation yields and the reduction of energy that is required to separate the volatile products. The paper addresses both of these challenges combining experimental innovations with a systems engineering approach. IBE is recovered from a gas-stripped fermenter whose potential for adsorption is researched and integrated with downstream options for separation. Design and integration is assisted using a systems approach that relies on mathematical models that regress and extrapolate experimental data for scale-up calculations. Process integration involves synthesis challenges to define biorefinery portfolios and systems integration to combine fermentation, stripping, adsorption, and distillation. The analysis considers 4 alternative biorefinery cases and presents results with significant savings in energy use and costs (up to 87% savings reported) after the application of energy integration to the IBE plant. Scenarios are analysed economically and confirm benefits in the use of adsorption and viable production yields.

Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of β-1,3- and β-1,4-linked β- d -glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli , and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum . Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum , were cloned separately into C. beijerinckii . To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 ( celA ) and pWUR4 ( celD ). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD ). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.

The unique chemical composition of seaweeds and their fast growth rates offer many opportunities for biorefining. In this article we argue that cascading biorefinery valorization concepts are viable alternatives to only using seaweeds as carbohydrate sources for the fermentative production of biofuels. However, many challenges remain with respect to use of seaweeds for chemical production, such as the large seasonal variation in the chemical composition of seaweeds.

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of alpha-MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH-mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.

Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia.

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack− strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack− and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (−50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia. Electronic supplementary material The online version of this article (doi:10.1007/s00253-011-3848-4) contains supplementary material, which is available to authorized users.

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum , for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum . The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans . CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli . The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli -produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.

Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and beta-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium.