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Summary: Volatile short-chain fatty acids such as butyric and acetic acid have been supplemented in poultry rations as a feed ingredient and nutritive supplement. A novel Clostridium fermentation solubles broth (MiruTyton), containing short-chain fatty acids, minerals, and amino acids, was supplemented in a standard corn/soybean meal–based ration at a rate of 750, 1,500, 3,000, and 6,000 mg/kg to achieve approximately 277, 554, 1,108, and 2,216 mg/kg of ammonium butyrate in the diet. After 6 wk, supplementation at all levels did not cause significant changes in the blood parameters or any live performance metric of mixed-sex Cobb-500 broilers (N = 1,920). In addition, administering 6,000 mg/kg of MiruTyton caused no significant lesions in any of the tissue examined except for an increase (P

The global demand for high-quality, protein-rich foods will continue to increase as the global population grows, along with income levels. Aquaculture is poised to help fulfill some of this demand, and is thus the fastest growing animal protein industry. A key challenge for it, though, is sourcing a sustainable, renewable protein ingredient. Single cell protein (SCP) products, protein meals based on microbial or algal biomass, have the potential to fulfill this need. Here, we review potential sources of SCP strains and their respective production processes, highlight recent advances on identification of new SCP strains and feedstocks, and, finally, review new feeding trial data on important aquaculture species, specifically Atlantic salmon, rainbow trout, and whiteleg shrimp.

Die industrielle Verwendung von C1-Gasen hat einerseits Tradition (z. B. Fischer-Tropsch-Katalyse), andererseits werden neue Ansatze erprobt und befinden sich z. T. an der Schwelle zur Kommerzialisierung. Kap. 16 stellt beispielhaft Entwicklungs-, Pilot-, Demonstrations- und Produktionsverfahren unterschiedlicher Unternehmen vor, darunter VITO aus Belgien, BASF, b.fab und Covestro aus Deutschland, AlgaTechnologies aus Israel, AlgaeParc und Photanol aus den Niederlanden sowie Cellana, Cyanotech, Lanzatech und White Dog Labs aus den USA.

Maximizing the conversion of biogenic carbon feedstocks into chemicals and fuels is essential for fermentation processes as feedstock costs and processing is commonly the greatest operating expense. Unfortunately, for most fermentations, over one-third of sugar carbon is lost to CO2 due to the decarboxylation of pyruvate to acetyl-CoA and limitations in the reducing power of the bio-feedstock. Here we show that anaerobic, non-photosynthetic mixotrophy, defined as the concurrent utilization of organic (for example, sugars) and inorganic (for example, CO2) substrates in a single organism, can overcome these constraints to increase product yields and reduce overall CO2 emissions. As a proof-of-concept, Clostridium ljungdahlii was engineered to produce acetone and achieved a mass yield 138% of the previous theoretical maximum using a high cell density continuous fermentation process. In addition, when enough reductant (that is, H2) is provided, the fermentation emits no CO2. Finally, we show that mixotrophy is a general trait among acetogens., Microbial fermentation yield is limited by CO2 loss in glycolysis. Here, the authors engineered Clostridium ljungdahlii for the anaerobic, non-photosynthetic mixotrophy production of acetone, increasing carbon product yield while reducing CO2 emissions from a biogenic feedstock fermentation.

Biological CO2 fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO2 fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (Blautia producta, Clostridium scatologenes and Thermoanaerobacter kivui) were chosen for further characterization, under a variety of conditions including H2- or syngas-fed sugar fermentations and a CO2-fed glycerol fermentation. In all cases, CO2 was fixed and carbon yields approached 100%. Finally, the model acetogen C. ljungdahlii was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO2 emissions.

In order to better characterize the initial stages of sporulation past Spo0A activation and the associated solventogenesis in the important industrial and model organism Clostridium acetobutylicum , the spoIIE gene was successfully disrupted and its expression was silenced. By silencing spoIIE , sporulation was blocked prior to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon plasmid-based complementation of spoIIE , sporulation was restored, although the number of spores formed was below that of the plasmid control strain. To investigate the impact of silencing spoIIE on the regulation of sporulation, transcript levels of sigF , sigE , and sigG were examined by semiquantitative reverse transcription-PCR, and the corresponding σ F , σ E , and σ G protein levels were determined by Western analysis. Expression of sigF was significantly reduced in the inactivation strain, and this resulted in very low σ F protein levels. Expression of sigE was barely detected, and no sigG transcript was detected at all; consequently, no σ E or σ G proteins were detected. These data suggest an autostimulatory role for σ F in C. acetobutylicum , in contrast to the model organism for endospore formation, Bacillus subtilis , and confirm that high-level expression of σ F is required for expression of σ E and σ G . Unlike the σ F and σ E inactivation strains, the SpoIIE inactivation strain did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both exponential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation—both stationary-phase events—remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only σ F but also the sigma factors σ E and σ G were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon ( spoIIAA - spoIIAB - sigF ) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ F expression was restored along with sporulation and σ E and σ G protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes ( csfB , gpr , spoIIP , sigG , lonB , and spoIIR ) that could be controlled by σ F , based on the Bacillus subtilis model, indicated that sigG may be under the control of σ F , but spoIIR , an important activator of σ E in B. subtilis , is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome—here, the C. acetobutylicum chromosome—with the aim of altering cell metabolism and differentiation.

Clostridium is a diverse genus of obligate anaerobic, endospore-forming, Gram-positive bacteria. Pathogenic species such as C. botulinum, C. difficile, C. perfringens, and C. tetani produce upwards of 18% of all known bacterial toxins, thus making Clostridium the “most toxic” prokaryotic genus (37) and a significant concern to human and animal health. Cellulolytic and solventogenic species, such as C. thermocellum, C. saccharobutylicum, C. cellulolyticum, and C. acetobutylicum are some of the best-studied biomass-degrading bacteria and exhibit significant potential for renewable biofuel and chemical production (22, 25, 26, 33). Additionally, nontoxigenic, proteolytic species, such as C. sporogenes and C. novyi, are being engineered into promising chemotherapeutic vehicles, in what is called clostridial-directed prodrug therapy (31). Despite the increased interest and activity in clostridia research, key fundamental questions regarding differentiation and physiology remain which are not only significant to the general understanding of the bacterium but also necessary for exploring the broad spectrum of clostridia applications. Arguably one of the most important fundamental questions is the genetic orchestration of clostridial sporulation and its coupling to other stationary-phase phenomena (22, 33, 34). For example, solvent formation (solventogenesis) is the characteristic stationary-phase phenomenon in solventogenic clostridia, and it is tightly if not causally associated with sporulation except in a set of genetically uncharacterized mutants obtained by random mutagenesis or continuous culture (15, 16, 23, 27). Recent comparative genomics approaches and DNA microarray analyses (17, 34) have reinforced the prevailing assumption that clostridial sporulation is similar if not identical to Bacillus subtilis sporulation, but experimentally this has recently been challenged (12). In B. subtilis, sporulation is initiated by a multicomponent phosphorelay that activates Spo0A in the predivisional cell (3), which then promotes the expression of mother cell-specific and prespore-specific sigma factors σE and σF (45). Active σE regulates the expression of numerous mother cell-specific genes (9) and, with the combined activity of SpoIIID, activates the mother cell-specific σK. The combined activities and intercompartmental communication of σE and σF lead to activation of the prespore-specific σG (45), which then regulates the expression of many prespore-specific genes (51). Disruption of sigF in B. subtilis blocked sporulation at stage II, resulting in normal-looking sporulation septum but also an accumulation of disporic cells (14). The sigE disruption also blocked sporulation at stage II, resulting in a similar, disporic morphology as the sigF mutant. Disruption of sigG blocked sporulation at stage III, exhibiting engulfment but no spore cortex or coat (7). σF activation is prevented until septation (41), when SpoIIE dephosphorylates the anti-anti-sigma factor SpoIIAA, which then binds the anti-sigma factor SpoIIAB to release active σF (41). σE activity is stalled until SpoIIGA processes the pro-σE, which requires septation and a physical interaction between SpoIIGA and the σF-regulated SpoIIR (20, 24, 36). Regulation of σG appears to be complex and multilayered (4, 28). Transcriptional analysis of pH-controlled C. acetobutylicum batch cultures suggested that the orchestration of Spo0A and the major sporulation sigma factors (σF, σE, and σG) are similar to those of B. subtilis (17). Sporulation factor activities were deduced by the expression patterns of putative regulon genes. The data suggested that Spo0A activity spikes during the transition from acidogenesis to solventogenesis (∼12 h after inoculation) and remains active throughout the duration of the culture. σF and σE both exhibited a major spike in activity during mid-stationary phase (24 h after inoculation), while σG activity spiked 6 h later, at 30 h after inoculation. Regarding the control of solventogenesis, relatively little is known beyond Spo0A activity (11, 38). Spo0A induces the expression of key solventogenic genes in C. acetobutylicum (11, 47), namely, the sol locus genes organized in two operons (aad-ctfA-ctfB and adc) located on the pSOL1 megaplasmid (6, 32). Furthermore, clostridia exhibit the unique clostridial cell form (16), which is an important sporulation-associated morphology. The clostridial cell form is characteristic of all clostridia, is commonly assumed to be the solvent-producing cell type in solventogenic clostridia (16, 49), and is characterized by the accumulation of granules of a glycogen-like (1,4-glucosyl glucan) biopolymer (39). Here we examined the roles of σE and σG in C. acetobutylicum by inactivating their genes and assessed their impact on solvent production and morphogenesis of the clostridial cell form. The disruption strains were characterized by Western blot analysis, Southern blot analysis, semiquantitative reverse transcription-PCR (RT-PCR), sporulation assays, phase-contrast microscopy, electron microscopy, flow cytometry (FC), and metabolite analysis. We found significant differences between these asporogenous clostridia disruption mutants and the corresponding B. subtilis mutants. Lastly, we examined the necessity of σE and σG for granulose formation and the development of the clostridial cell form, as well as for solvent formation.

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum . Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

Mass yields of biofuels and chemicals from sugar fermentations are limited by the decarboxylation reactions involved in Embden-Meyerhof-Parnas (EMP) glycolysis. This paper reviews one route to recapture evolved CO2 using the Wood-Ljungdahl carbon fixation pathway (WLP) in a process called anaerobic, non-photosynthetic (ANP) mixotrophic fermentation. In ANP mixotrophic fermentation, the two molecules of CO2 and eight electrons produced from glycolysis are used by the WLP to generate three molecules of acetyl-CoA from glucose, rather than the two molecules that are produced by typical fermentation processes. In this review, we define the bounds of ANP mixotrophy, calculate the potential metabolic advantages, and discuss the viability in a number of host organisms. Additionally, we highlight recent accomplishments in the field, including the recent discovery of electron bifurcation in acetogens, and close with recommendations to realize mixotrophic biofuel and biochemical production.

In order to better characterize the initial stages of sporulation past Spo0A activation and the associated solventogenesis in the important industrial and model organism Clostridium acetobutylicum, the spoIIE gene was successfully disrupted and its expression was silenced. By silencing spoIIE, sporulation was blocked prior to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon plasmid-based complementation of spoIIE, sporulation was restored, although the number of spores formed was below that of the plasmid control strain. To investigate the impact of silencing spoIIE on the regulation of sporulation, transcript levels of sigF, sigE, and sigG were examined by semiquantitative reverse transcription-PCR, and the corresponding σF, σE, and σG protein levels were determined by Western analysis. Expression of sigF was significantly reduced in the inactivation strain, and this resulted in very low σF protein levels. Expression of sigE was barely detected, and no sigG transcript was detected at all; consequently, no σE or σG proteins were detected. These data suggest an autostimulatory role for σF in C. acetobutylicum, in contrast to the model organism for endospore formation, Bacillus subtilis, and confirm that high-level expression of σF is required for expression of σE and σG. Unlike the σF and σE inactivation strains, the SpoIIE inactivation strain did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both exponential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation--both stationary-phase events--remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only σ(F) but also the sigma factors σ(E) and σ(G) were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon (spoIIAA-spoIIAB-sigF) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ(F) expression was restored along with sporulation and σ(E) and σ(G) protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (csfB, gpr, spoIIP, sigG, lonB, and spoIIR) that could be controlled by σ(F), based on the Bacillus subtilis model, indicated that sigG may be under the control of σ(F), but spoIIR, an important activator of σ(E) in B. subtilis, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome--here, the C. acetobutylicum chromosome--with the aim of altering cell metabolism and differentiation.

Central to all clostridia is the orchestration of endospore formation (i.e., sporulation) and, specifically, the roles of differentiation-associated sigma factors. Moreover, there is considerable applied interest in understanding the roles of these sigma factors in other stationary-phase phenomena, such as solvent production (i.e., solventogenesis). Here we separately inactivated by gene disruption the major sporulation-specific sigma factors, σ(E) and σ(G), and performed an initial analysis to elucidate their roles in sporulation-related morphogenesis and solventogenesis in Clostridium acetobutylicum. The terminal differentiation phenotype for the sigE inactivation mutant stalled in sporulation prior to asymmetric septum formation, appeared vegetative-like often with an accumulation of DNA at both poles, frequently exhibited two longitudinal internal membranes, and did not synthesize granulose. The sigE inactivation mutant did produce the characteristic solvents (i.e., butanol and acetone), but the extent of solventogenesis was dependent on the physiological state of the inoculum. The sigG inactivation mutant stalled in sporulation during endospore maturation, exhibiting engulfment and partial cortex and spore coat formation. Lastly, the sigG inactivation mutant did produce granulose and exhibited wild-type-like solventogenesis.

Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering. We demonstrate the importance of FC for elucidating population heterogeneity because of developmental processes or epigenetic regulation. FC can be engaged for both synthetic and analytical applications of complex phenotypes within a single species, multispecies, and microbial-library populations. Examples include methods to identify developmental microbial stages associated with productive metabolic phenotypes, select desirable promoters from a single species or metagenomic libraries, and to screen designer riboswitches for synthetic-biology applications.

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.

A detailed microarray analysis of transcription during sporulation of the strict anaerobe and endospore former Clostridium acetobutylicum is presented., Background Clostridia are ancient soil organisms of major importance to human and animal health and physiology, cellulose degradation, and the production of biofuels from renewable resources. Elucidation of their sporulation program is critical for understanding important clostridial programs pertaining to their physiology and their industrial or environmental applications. Results Using a sensitive DNA-microarray platform and 25 sampling timepoints, we reveal the genome-scale transcriptional basis of the Clostridium acetobutylicum sporulation program carried deep into stationary phase. A significant fraction of the genes displayed temporal expression in six distinct clusters of expression, which were analyzed with assistance from ontological classifications in order to illuminate all known physiological observations and differentiation stages of this industrial organism. The dynamic orchestration of all known sporulation sigma factors was investigated, whereby in addition to their transcriptional profiles, both in terms of intensity and differential expression, their activity was assessed by the average transcriptional patterns of putative canonical genes of their regulon. All sigma factors of unknown function were investigated by combining transcriptional data with predicted promoter binding motifs and antisense-RNA downregulation to provide a preliminary assessment of their roles in sporulation. Downregulation of two of these sigma factors, CAC1766 and CAP0167, affected the developmental process of sporulation and are apparently novel sporulation-related sigma factors. Conclusion This is the first detailed roadmap of clostridial sporulation, the most detailed transcriptional study ever reported for a strict anaerobe and endospore former, and the first reported holistic effort to illuminate cellular physiology and differentiation of a lesser known organism.