Glycerol-driven adaptive evolution for the production of low-molecular-weight Welan gum: Characterization and activity evaluation.

Through adaptive laboratory evolution (ALE) of Sphingomonas sp. ATCC 31555, fermentation for production of low-molecular-weight welan gum (LMW-WG) was performed using glycerol as sole carbon source. During ALE, GPC-MALS analysis revealed a gradual decrease in WG molecular weight with the increase of adaptation cycles, accompanied by changes in solution conformation. LMW-WG was purified and structurally analyzed using GPC-MALS, monosaccharide composition analysis, infrared spectroscopy, NMR analysis, atomic force microscopy, and scanning electron microscopy. Subsequently, LMW-WG obtains hydration, transparency, antioxidant activity, and rheological properties. Finally, an in vitro simulation colon reactor was used to evaluate potential prebiotic properties of LMW-WG as dietary fiber. Compared with WG produced using sucrose as substrate, LMW-WG exhibited a fourfold reduction in molecular weight while maintaining moderate viscosity. Structurally, L-Rha nearly completely replaced L-Man. Furthermore, LMW-WG demonstrated excellent hydration, antioxidant activity, and high transparency. It also exhibited resistance to saliva and gastrointestinal digestion, showcasing a favorable colonization effect on Bifidobacterium, making it a promising symbiotic agent.

Engineering and transcriptome study of Saccharomyces cerevisiae to produce ginsenoside compound K by glycerol.

Synthetic biology-based engineering of Saccharomyces cerevisiae to produce terpenoid natural products is an effective strategy for their industrial application. Previously, we observed that glycerol addition was beneficial for ginsenoside compound K (CK) production in a S. cerevisiae when it was fermented using the YPD medium. Here, we reconstructed the CK synthesis and glycerol catabolic pathway in a high-yield protopanaxadiol (PPD) S. cerevisiae strain. Remarkably, our engineered strain exhibited the ability to utilize glycerol as the sole carbon source, resulting in a significantly enhanced production of 433.1 ± 8.3 mg L-1 of CK, which was 2.4 times higher compared to that obtained in glucose medium. Transcriptomic analysis revealed that the transcript levels of several key genes involved in the mevalonate (MVA) pathway and the uridine diphosphate glucose (UDPG) synthesis pathway were up-regulated in response to glycerol. The addition of glycerol enhanced CK titers by augmenting the flux of the terpene synthesis pathway and facilitating the production of glycosyl donors. These results suggest that glycerol is a promising carbon source in S. cerevisiae, especially for the production of triterpenoid saponins.

Heterologous 1,3-Propanediol Production Using Different Recombinant Clostridium beijerinckii DSM 6423 Strains.

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_Ppta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach.

Assessing main process mechanism and rates of sulfate reduction by granular biomass fed with glycerol under sulfidogenic conditions.

Sulfate-reducing bioreactors for sulfide production are the initial stage of processes targeting elemental sulfur recovery from sulfate-rich effluents. In this work, the principal reactions involved in glycerol fermentation and sulfate reduction using glycerol and its fermentation products as electron donors were assessed together with their specific consumption/production rates. A battery of batch activity tests with and without sulfate were performed with glycerol and with each fermentation product using a non-methanogenic but sulfidogenic granular sludge from an up-flow anaerobic sludge blanket (UASB) reactor operated under long-term while fed with crude glycerol. As a result, a mechanistic approach based on the experimental observations is proposed in this work. Glycerol was mainly fermented to 1,3-propanediol, ethanol, formate, propionate and acetate by fermentative bacteria. All organic intermediates were found to be further used by sulfate reducing bacteria (SRB) for sulfate reduction except for acetate. The most abundant genus detected under sulfidogenic conditions were Propionispora (15.2%), Dysgonomonas (13.2%), Desulfobulbus (11.6%) and Desulfovibrio (10.8%). The last two SRB genera accounted for 22.4% of the total amount of retrieved sequences, which were probably performing an incomplete oxidation of the carbon source in the sulfidogenic UASB reactor. As single substrates, specific sulfate reduction rates (SRRs) using low molecular weight (MW) carbon sources (formate and ethanol) were 39% higher than those using high-MW ones (propionate, 1,3-propanediol and butanol). However, SRRs in glycerol-fed tests showed that 1,3-propanediol played a major role in sulfate reduction in addition to formate and ethanol.

An Escherichia coli FdrA Variant Derived from Syntrophic Coculture with a Methanogen Increases Succinate Production Due to Changes in Allantoin Degradation.

Wild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrAD296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrAD296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production. IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrAD296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the "orphan enzyme" oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.

Propionic Acid: Method of Production, Current State and Perspectives.

During the past years, there has been a growing interest in the bioproduction of propionic acid by Propionibacterium. One of the major limitations of the existing models lies in their low productivity yield. Hence, many strategies have been proposed in order to circumvent this obstacle. This article provides a comprehensive synthesis and review of important biotechnological aspects of propionic acid production as a common ingredient in food and biotechnology industries. We first discuss some of the most important production processes, mainly focusing on biological production. Then, we provide a summary of important propionic acid producers, including Propionibacterium freudenreichii and Propionibacterium acidipropionici, as well as a wide range of reported growth/production media. Furthermore, we describe bioprocess variables that can have impact on the production yield. Finally, we propose methods for the extraction and analysis of propionic acid and put forward strategies for overcoming the limitations of competitive microbial production from the economical point of view. Several factors influence the propionic acid concentration and productivity such as culture conditions, type and bioreactor scale; however, the pH value and temperature are the most important ones. Given that there are many reports about propionic acid production from glucose, whey permeate, glycerol, lactic acid, hemicelluloses, hydrolyzed corn meal, lactose, sugarcane molasses and enzymatically hydrolyzed whole wheat flour, only few review articles evaluate biotechnological aspects, i.e. bioprocess variables.

Influence of C4 -Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase-grown fermenting Escherichia coli.

During mixed-acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH-H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH-H activities during glycerol but not glucose fermentation at acidic pH. During glycerol fermentation at pH 5.5, inactivation of either one or all Dcu carriers increased total Hyd activity by 60% compared with wild type. Under the same growth conditions, a dcuACBD mutant had a twofold higher FDH-H activity. When glucose was fermented in dcuD single mutant at pH 5.5, the FDH-H activity was also increased twofold compared with wild type. Interestingly, in dcuD or dcuACBD mutants at pH 7.5, Hyd activity was lowered by 20%. Taken together, it can be concluded that during glucose fermentation at pH 7.5, lack of DcuD affects Hyd enzyme activity, but at pH 5.5, it has a stronger effect on FDH-H activity. During glycerol fermentation, lack of Dcu carriers increased Hyd and FDH-H activities as revealed at pH 5.5. The results suggest that impairing Dcu transport function increases intracellular formate levels and thus affects H2 cycling and proton-motive force generation.

Shaping microbial consortia in coupling glycerol fermentation and carboxylate chain elongation for Co-production of 1,3-propanediol and caproate: Pathways and mechanisms.

Glycerol is presently being generated in surplus with the rapid growth of the biodiesel industry and seeks ways to be upcycled, rather than to be treated with costs. Glycerol for the co-production of 1,3-propanediol (1,3-PDO) and caproate has a great prospect. Yet, its technical difficulty lies in the enhancement of caproate productivity, which requires the presence of ethanol as a co-substrate and necessitates the co-existence of functional microbes for glycerol fermentation and chain elongation. This study successfully achieved 6.38 mM C 1,3-PDO d-1 and 2.95 mM C caproate d-1 in a 2-L mixed-cultured semi-continuous fermenter with a glycerol-ethanol-acetate stoichiometric ratio of 4:3:1. Such conversions were mainly facilitated by a microbial community of Eubacterium limosum, Clostridium kluyveri and Massilibacterium senegalense. With such a synergistic microbiome, the co-production of 1,3-PDO and caproate was achieved from glycerol without ethanol addition. Based on metagenomics, E. limosum is capable of converting glycerol to 1,3-PDO, ethanol and H2, and also redirecting the electron potential of H2 into acetate via the Wood-Ljungdahl pathway, which is then used for chain elongation. C. kluyveri worked synergistically with E. limosum by consuming ethanol and acetate for caproate production. M. senegalense encodes for ethanol oxidation to acetate and butyrate, facilitating the generation of these intermediates for C. kluyveri elongation to caproate. During the transition between fermentation and elongation, an unexpected observation of poly-ß-hydroxybutyrate (PHB) formation and reutilization by M. senegalense may be associated with butyrate formation for further caproate generation. The knowledge gleaned from the substrate constitute, microbial consortium and their synergetic metabolism demonstrates a resource upgrade potential for crude glycerol or glycerol-containing wastewater generated from the biodiesel industry.

Heterologous expression of the human Phosphoenol Pyruvate Carboxykinase (hPEPCK-M) improves hydrogen and ethanol synthesis in the Escherichia coli dcuD mutant when grown in a glycerol-based medium.

The production of biodiesel has emerged as an alternative to fossil fuels. However, this industry generates glycerol as a by-product in such large quantities that it has become an environmental problem. The biotransformation of this excess glycerol into other renewable bio-energy sources, like H2 and ethanol, by microorganisms such as Escherichia coli is an interesting possibility that warrants investigation. In this work we hypothesized that the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) could be improved by a controlled expression of the human mitochondrial GTP-dependent PEP carboxykinase. This heterologous expression was tested in several E. coli mutant backgrounds with increased availability of C4 intermediates. It was found that this metabolic rewiring improved the synthesis of the target products in several mutants, with the dcuD mutant being the most suitable background for hydrogen and ethanol specific productions and glycerol consumption. These factors increased by 2.46, 1.73 and 1.95 times, respectively, when compared to those obtained for the wild-type strain.

Anaerobes in Industrial- and Environmental Biotechnology.

Anaerobic microorganisms present in diverse ecological niches employ alternative strategies for energy conservation in the absence of oxygen which enables them to play a key role in maintaining the global cycles of carbon, nitrogen, and sulfur, and the breakdown of persistent compounds. Thereby they become useful tools in industrial and environmental biotechnology. Although anaerobes have been relatively neglected in comparison to their aerobic counterparts, with increasing knowledge about their diversity and metabolic potential and the development of genetic tools and process technologies to utilize them, we now see a rapid expansion of their applications in the society. This chapter summarizes some of the developments in the use of anaerobes as tools for biomass valorization, in production of energy carriers and chemicals, wastewater treatment, and the strong potential in soil remediation. The ability of several autotrophic anaerobes to reduce carbon dioxide is attracting growing attention as a means for developing a platform for conversion of waste gases to chemicals, materials, and biofuels.

Continuous production of n-butanol by Clostridium pasteurianum DSM 525 using suspended and surface-immobilized cells.

For n-butanol production by Clostridium pasteurianum DSM 525, a modified reinforced Clostridium medium was used, where glucose was alternated with glycerol and two kinds of continuous fermentation were tested using suspended and surface immobilized cells on corn stover pieces. A steady state, with butanol productivity of 4.2g/Lh, was reached during the packed-bed continuous fermentation at a dilution rate of 0.44h(-1). The average n-butanol concentration, yield and the ratio of n-butanol/liquid by-products were 10.4g/L, 33 % and 2.5, respectively. Unexpectedly, during continuous fermentation with suspended cells, at a dilution rate of 0.01h(-1), steady-state was not achieved and regular oscillations occurred in all measured variables, i.e. concentrations of glycerol, products and the number of cells stained with the fluorescent dyes carboxy fluorescein diacetate and propidium iodide. A possible explanation for oscillatory/steady-state behavior of suspended/surface-attached cells, respectively, may be specific butanol toxicity (toxicity per cell), which was higher/lower in respective cases, and which might be caused by lower/higher cell numbers respectively in both systems.

Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption.

Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

Isolation of a solventogenic Clostridium sp. strain: fermentation of glycerol to n-butanol, analysis of the bcs operon region and its potential regulatory elements.

A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474(T). GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v). The solventogenesis genes crt, bcd, etfA/B and hbd composing the bcs (butyryl-CoA synthesis) operon of C. tetanomorphum GT6 were sequenced. They occur in a genomic arrangement identical to those in other solventogenic clostridia. Furthermore, the sequence of a potential regulator gene highly similar to that of the NADH-sensing Rex family of regulatory genes was found upstream of the bcs operon. Potential binding sites for Rex have been identified in the promoter region of the bcs operon of solvent producing clostridia as well as upstream of other genes involved in NADH oxidation. This indicates a fundamental role of Rex in the regulation of fermentation products in anaerobic, and especially in solventogenic bacteria.

Escherichia coli multiple [Ni-Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs.

Escherichia coli evolves H2 via multiple [Ni-Fe]-hydrogenases (Hyd). This activity under hyper- and hypo-osmotic stress was investigated with mutants lacking different Hyd enzymes during glycerol fermentation. Inhibitory effects of hypo-stress on H2 production was stronger at pH 6.5 in wild type and mutants except fhlA, which encodes a transcriptional activator for Hyd-3, compared with the effects of N,N'-dicyclohexylcarbodiimide. These results indicate that Hyd-3 and Hyd-4 are osmosensitive at pH 7.5. Hyd-4 and FhlA are implicated in osmotic stress response at pH 6.5. Hyd-1 and FhlA might be osmosensitive at pH 5.5. Thus, osmosensitivity of Hyd enzymes is a novel property that depends on pH. This is significant for mechanisms of cell osmoregulation and H2 production biotechnology when glycerol is used as a fermentation substrate.

Mechanism of Corn Steep Liquor during Glycerol Fermentation by Candida glycerinogenes / 微生物学通报

Using chemically defined medium as the control, mechanism of corn steep liquor (CSL) in complex medium during glycerol production by Candida glycerinogenes was studied.The results showed that there were three key factors in CSL that had some great influences on glycerol fermentation of C.glycerinogenes, including phosphorus, nitrogen, and trace elements.The maximum glycerol yield of 53.44% was achieved at an optimal phosphorus concentration of 121.75mg/L, where the CSL concentration was 14g/L.Phosphorus in CSL could control the distribution of carbon metabolism flux between EMP pathway and HMP pathway.With the increase in CSL concentrations, superfluous phosphorus could restrain HMP pathway and activate EMP pathway, thus resulting in remarkable changes in various fermentation parameters of complex medium.Nitrogen in CSL could play a cooperative role in the regulative function of phosphorus.However, it was not a suitable nitrogen source for C.glycerinogenes.Trace elements in CSL could markedly improve the glucose consumption rate, accelerate the cell growth, and enhance the glycerol yield.