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.
► Ulva lactuca was characterized as feedstock for the acetone, butanol and ethanol fermentation. ► Hydrolysates were obtained using mild pretreatment conditions and commercial cellulases. ► Ulva ...lactuca hydrolysate was used as substrate for fermentation by two different strains. ► Rhamnose was utilized by C. beijerinckii for production of 1,2-propanediol.
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 (0.35g 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 (9.7g/L).
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.
•Gas stripping of isopropanol, butanol and ethanol (IBE) from model solutions was characterized.•Solvent removal during C. beijerinckii cultivation enhances IBE productivity.•New repeated batch ...process with discontinuous solvent removal at 70°C has been demonstrated.
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.
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 3.6kb. 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.
•A two-plasmid inducible CRISPR/Cas9 genome editing tool has been developed•Editing efficiency of 100% was achieved in Clostridium acetobutylicum ATCC 824•Nucleotide substitution, deletion and integrations up to 3.6kb were performed
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.
Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but ...also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.
SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ
, during sporulation. A Δ
mutant of
NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate ...but still produced granulose and solvents. Microscopy analysis also showed that the cells of the Δ
mutant are elongated with the presence of multiple septa. This observation suggests that in
, SpoIIE is necessary for the completion of the sporulation process, as seen in
and
. Moreover, when grown in reactors, the
mutant produced higher levels of solvents than the wild type strain. The impact of the
inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ
, σ
, σ
, σ
) were strongly down-regulated in the mutant. Inactivation of
also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in
.
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
.
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