Clostridium autoethanogenum protein as an alternative for soybean meal in the diet of rainbow trout (Oncorhynchus mykiss): Considering the effects of microcrystalline cellulose

Impact of acetogenic partner and pH on syngas fermentation in Clostridium kluyveri co-cultures.

Simultaneous conversion of syngas and sugars is a promising approach to overcome limitations of syngas fermentation. Clostridium autoethanogenum LAbrini, obtained by adaptive laboratory evolution, is known to show improved autotrophic process performance. Under purely autotrophic conditions, C. autoethanogenum LAbrini exhibits substantially faster growth and biomass formation compared to the wild-type in fully controlled, stirred-tank bioreactors with a continuous gas supply. In mixotrophic processes, the pre-culture strategy has a significant impact on the growth and metabolic activity of C. autoethanogenum LAbrini. C. autoethanogenum LAbrini can metabolize sugars (D-fructose, D-xylose, or L-arabinose) and CO simultaneously. All mixotrophic batch processes showed increased growth and product formation compared to the autotrophic process. The mixotrophic batch process with D-fructose enabled superior production of alcohols (10.7 g L−1 ethanol and 3.2 g L−1 D-2,3-butanediol) with a heterotrophic pre-culture. Using an autotrophic pre-culture and L-arabinose resulted in a total alcohol formation of more than 13 g L−1. The formation of meso-2,3-butanediol (>0.50 g L−1) occurred exclusively under mixotrophic conditions. Thus, C. autoethanogenum LAbrini, clearly representing notable improvements over the wild-type strain in mixotrophic batch processes, offers a good basis for further strain improvements to shift the product range even further towards more reduced products.

A 10-week growth experiment was conducted to evaluate the physiological effects of dietary phosphorus supplementation on red swamp crayfish (Procambarus clarkii) feeding diets with high Clostridium autoethanogenum protein (CAP) levels. Six isonitrogenous and isolipid diets were formulated: The FM diet contained 10% fishmeal, which is equivalent to a dietary phosphorus level of 1.41%, and the CAP, CAPSP1, CAPSP2, and CAPSP3 diets substituted all fishmeal with CAP and supplemented with 0, 2.5%, 3%, and 3.5% Ca(H2PO4)2, respectively (corresponding to dietary phosphorus levels of 0.66%, 1.27%, 1.40%, and 1.52%). A total of 600 crayfish with an initial mean weight of (5.01 ± 0.02) g were selected and randomly assigned to 15 cages for feeding and sampled at the end of the experiment. Results indicate that high-dose CAP replacing fishmeal caused abnormal hepatopancreatic tissue structure in crayfish, exacerbating lipid deposition and oxidative stress. Compared with the CAP group, the specific growth rate (SGR) of crayfish in the CAPSP2 and CAPSP3 groups significantly increased (p < 0.05). The activities of antioxidant enzymes and lipid-degrading enzymes in the hepatopancreas, along with the relative expression of related genes, were significantly enhanced (p < 0.05). Metabolomic analysis demonstrated significant differences in major differential metabolites and metabolic pathways between the CAP group crayfish and the CAPSP2 group (p < 0.05). CAPSP2 group crayfish exhibited a higher content of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC), with significant enrichment in glycerophospholipid metabolism and fatty acid metabolism pathways (p < 0.05). Overall, supplementing dietary phosphorus levels to 1.40–1.52% effectively mitigated growth retardation, oxidative damage, and lipid metabolism disorders induced by high-proportion CAP replacement of fishmeal.

Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.

Evaluation of Clostridium autoethanogenum protein as a fish meal substitute in diets for juvenile mud crab (Scylla paramamosain)

Methylene-tetrahydrofolate reductase (MTHFR) is an important enzyme for acetogenic carbon fixation, but the redox mechanism driving this reaction is not clearly understood. Previous enzymology work and energetic accounting on species such as Clostridium autoethanogenum has led to confounding results when placed in the context of in vivo experiments. In this work, we create multiple C. autoethanogenum strains harboring alternative MTHFR enzyme complexes as well as genome-scale metabolic models to better understand how these organisms conserve energy on gas substrates. The inclusion of a Type-III MTHFR unexpectedly allows for higher growth than expected and suggests the possibility of an additional redox balancing cycle employed during autotrophic growth.

Mineral supplementation protected intestinal health in Litopenaeus vannamei by alleviating the immune response and oxidative damage caused by high-level of Clostridium autoethanogenum protein

Effects of fish meal replacement by Clostridium autoethanogenum protein on growth performance, serum biochemistry, antioxidant capacity, immune responses and muscle quality in black carp (Mylopharyngodon piceus)

ABSTRACTGas‐fermenting acetogens, such as Clostridium autoethanogenum, have emerged as promising biocatalysts capable of converting CO and CO2‐containing gases into fuels and chemicals relevant for a circular economy. However, the functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in C. autoethanogenum. In this study, we deleted one of the targets—CLAU_0471 (proposed amino acid permease)—with high mutation occurrence in ALE isolates and extensively characterised the autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterised two previously reverse‐engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two‐component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g., low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3‐butanediol production, while RE1 performed similarly to the best‐performing previously characterised ALE isolate LAbrini. The three reverse‐engineered strains showed similarities in proteome expression, and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype–phenotype relationships for a better understanding of the metabolism of an industrially relevant acetogen.

Salinity is a critical environmental factor in aquaculture, and Clostridium autoethanogenum protein (CAP) shows potential as a new feed protein source. This 8-week study compared two diets (fish meal [FM] and CAP) for juvenile Pacific white shrimp (Litopenaeus vannamei) at salinity levels of 15‰, 30‰, and 45‰. A total of 720 L. vannamei with an initial average body weight of 0.38 ± 0.01 g were randomly assigned to six experimental groups, each with three biological replicates of 40 shrimp per replicate. The study examined CAP's impact on shrimp growth, immune response, and transcriptome, using two-way ANOVA to analyze the results. The results indicated that compared to the FM group, the shrimp in the CAP group exhibited a significantly higher weight gain rate and specific growth rate at the same salinity (P < 0.05). However, at 45‰ salinity, the shrimp in the CAP group had a higher feed conversion ratio and feed intake compared to the FM group (P < 0.05). After infection with white spot syndrome virus, the CAP group exhibited a significantly higher survival rate at 15‰ and 45‰ salinity compared to the FM group (P < 0.05). As salinity increased, most immune enzyme activities and gene expression levels in the FM group initially increased and then decreased (P < 0.05). Under the same salinity, except for phenoloxidase activity which showed no significant difference at 45‰ salinity, all other immune-related indicators and gene expressions in the CAP group were significantly higher than those in the FM group (P < 0.05). Transcriptome analysis revealed that the differentially expressed genes (DEGs) between FM and CAP groups at various salinity levels were primarily associated with immune and metabolic pathways. Additionally, by combining the analysis of these DEGs with immune-related indicators, it was observed that under different salinity conditions, CAP was associated with the co-expression of immune and metabolism-related genes as well as changes in enzyme activity. In summary, CAP as the main protein source boosts growth, disease resistance, and nonspecific immunity in L. vannamei, while also regulating immune enzyme activity and gene expression to improve adaptability to salinity changes.

Evaluating the inclusion of Clostridium autoethanogenum protein instead of fishmeal protein in diets for European seabass (Dicentrarchus labrax): Growth performance, digestive enzymes, health status, and tissues investigations

Fluorescent protein-based anaerobic reporter for construction of promoter libraries in Clostridium autoethanogenum.

Two-stage continuous CO fermentation process strategy for high-titer bioethanol production using Clostridium autoethanogenum.

Combined microbiomic and transcriptomic analysis revealed that dietary Clostridium autoethanogenum protein could improve the disease resistance of Litopenaeus vannamei by regulating the oxidative phosphorylation

Abstract This study investigated the heterotrophic growth kinetics of Clostridium autoethanogenum in batch cultures using xylose, a hemicellulose hydrolysis product, as the fermentation substrate. The growth data were modeled using the Monod equation. No substrate inhibition was observed within the tested range of xylose concentrations. However, microbial growth deviated from standard Monod kinetics, with distinct growth behavior at different xylose concentration ranges. These findings suggest that C. autoethanogenum utilizes two distinct substrate uptake systems: one with high affinity for xylose and another with lower affinity.

A novel experimental method to determine substrate uptake kinetics of gas-fermenting microorganisms applied to carbon monoxide-fermenting Clostridium autoethanogenum

Integrated metabolomics and proteomics analysis to provide insights into muscle atrophy of turbot Scophthalmus maximus by dietary Clostridium autoethanogenum protein

Interaction of dietary replacements of fishmeal by protein blend and feeding frequency on growth performance and protein utilization of gibel carp (Carassius gibelio var. CAS Ⅴ)

In the ancient microbial Wood-Ljungdahl pathway, carbon dioxide (CO2) is fixed in a multistep process that ends with acetyl-coenzyme A (acetyl-CoA) synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). In this work, we present structural snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction, including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site, and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer, and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme, which is crucial for CO2 fixation in anaerobic organisms.