Clostridium Diolis Research Articles

Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.

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.

Draft Genome Sequence of Clostridium beijerinckii Strain mbf-VZ-132, Isolated from an Environmental Soil Sample.

Clostridium beijerinckii strain mbf-VZ-132 was isolated from soil in Freising-Weihenstephan (Bavaria, Germany). The 16S rRNA gene sequence showed a 99.9% sequence identity to that of Clostridium diolis DSM 15410, which was recently reclassified as C. beijerinckii In this study, we present the draft genome sequence of C. beijerinckii mbf-VZ-132 based on PacBio sequencing.

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable.Graphical abstract

Developing Clostridium diolis as a biorefinery chassis by genetic manipulation

Clostridium diolis can efficiently utilize various inexpensive, renewable resources such as crude glycerol and lignocellulosic biomass hydrolysate to produce bulk chemicals and fuels. However, its study has been impeded by the lack of efficient plasmids electro-transformation techniques. In this study, an efficient electroporation protocol for C. diolis was developed and two replicons functional in C. diolis were identified. After optimizing parameters, the electro-transformation efficiency was enhanced from 5 to 692 transformants/ug DNA. Moreover, metabolic engineering of C. diolis was performed as proof of concept for the first time. By simply overexpressing heterologous genes based on the replicable plasmids, the strain was engineered to improve productions of diol (1,3-propanediol) and n-alcohol (butanol), and to enable butyl acetate synthesis in vivo, respectively under different culture conditions. This work represented a milestone of breeding C. diolis using metabolic engineering, and paved the way for studying C. diolis on the molecular level.

Reclassification of Clostridium diolis Biebl and Spröer 2003 as a later heterotypic synonym of Clostridium beijerinckii Donker 1926 (Approved Lists 1980) emend. Keis et al. 2001.

Clostridium diolis shares high similarity based on 16S rRNA gene sequences and fatty acid composition with Clostridium beijerinckii. In this study, the taxonomic status of C. diolis was clarified using genomic and phenotypic approaches. High similarity was detected among C. diolis DSM 15410T, C. beijerinckii DSM 791T and NCTC 13035T, showing average nucleotide identity on blast and in silico DNA-DNA hybridization values over 97 and 85 %, respectively. Results of investigations for substrate utilization and enzyme activity displayed no striking differences between C. diolis DSM 15410T and C. beijerinckii JCM 1390T. Based on the results, we propose the reclassification of Clostridium diolis as a later heterotypic synonym of Clostridium beijerinckii. The type strain is ATCC 25752T (=CIP 104308T=DSM 791T=JCM 1390T=LMG 5716T=NCTC 13035T).

PRODUCCIÓN DE 1,3-PROPANODIOL A PARTIR DE GLICEROL CRUDO: DISEÑO DEL BIOPROCESO Y ANÁLISIS DE RENTABILIDAD

Crude glycerol is the major byproduct of the biodiesel industry, and its low price due to its abundant market supply impacts negatively on this industry. The microbial production of 1,3-propanediol (1,3-PD) is a very promising way for valorizing glycerol. Clostridium diolis DSM 15410 converts glycerol into 1,3-PD and is of industrial value due to its high fermentation yields and productivities. The aim of this study was to design a biotechnological process for the continuous production of 1,3-PD (> 99.0% purity) from crude glycerol using C. diolis DSM 15410 as biological system, and evaluate the process economic profitability under different scenarios. Optimal fermentation parameters were considered for the analysis. The effects of the selling price of 1,3-PD ($2.3-$6/t), the purchase cost of crude glycerol ($100-$300/t) and the process throughput (100-1,000 kg 1,3-PD/h) on the process profitability were studied. The results showed that process is economically profitable over a wide range of scenario combinations. This paper serves as a means for strategic decision making (e.g., go/no-go investment decisions) and further optimization of the 1,3-PD production process from crude glycerol.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.

The Draft Genome Sequence of Clostridium beijerinckii NJP7, a Unique Bacterium Capable of Producing Isopropanol-Butanol from Hemicellulose Through Consolidated Bioprocessing.

A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

Optimization of conditions for direct bio-hydrogen production from water hyacinth by clostridium diolis C32-KKU

Water hyacinth contains cellulose and hemicellulose which can be used as a substrate for bio-hydrogen production. Clostridium diolis C32-KKU, a cellulolytic bacterium, was employed to directly ferment water hyacinth to bio-hydrogen. The objective of this study was to optimize the direct bio-hydrogen production from water hyacinth by C. diolis C32-KKU. Two operation modes for bio-hydrogen production i.e. static and shaking modes were investigated. The results showed that the shaking mode was more effective than the static mode for hydrogen production. The shaking mode was then used to optimize bio-hydrogen production by variation of initial pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0) and initial water hyacinth concentration (5, 10, 15, 20, 25 and 30 g-dry weight (dw)/L). The maximum hydrogen production of 19 mL/L was obtained at the initial pH of 5.5 and water hyacinth concentration of 10 g-dw/L. The cellulase activity of 0.0081 unit/mL was obtained under the optimal condition. The results of this study showed that direct bio-hydrogen production from lignocellulosic materials could be feasible.

Co-utilization of glycerol and lignocellulosic hydrolysates enhances anaerobic 1,3-propanediol production by Clostridium diolis.

Anaerobic fermentation using lignocellulosic hydrolysates as co-substrates is an economically attractive method to enhance 1,3-propanediol (1,3-PD) production by increasing the conversion yield from glycerol. Lignocellulosic hydrolysates contain the mixed sugars that are primarily glucose, xylose, and arabinose. Therefore, these three individual sugars were used, separately, as co-substrates with glycerol, in 1,3-PD production by a Clostridium diolis strain DSM 15410, resulting in an 18%–28% increase in the 1,3-PD yield. Co-fermentation of the mixed sugars and glycerol obtained a higher intracellular NADH/NAD+ ratio and increased the 1,3-PD yield by 22% relative to fermentation of glycerol alone. Thereafter, two kinds of lignocellulosic hydrolysates, corn stover hydrolysate and corncob molasses, were individually co-fermented with glycerol. The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol. Fed-batch co-fermentation was also performed, improving the 1,3-PD yield (from 0.62 mol/mol to 0.82 mol/mol). These results demonstrate that the co-fermentation strategy is an efficient and economical way to produce 1,3-PD from glycerol.

Recycling of spent oil bleaching earth as source of glycerol for the anaerobic production of acetone, butanol, and ethanol with Clostridium diolis and lipolytic Clostridium lundense

A major part of edible oil is subjected to bleaching procedures, primarily with minerals applied as adsorbers. Their recycling is currently done either by regaining the oil via organic solvent extraction or by using the spent bleaching earth (SBE) as additive for animal feed, etc. As a new method, the reutilization of the by‐product SBE for the microbiologic formation of acetone, butanol, and ethanol (ABE) is presented as proof‐of‐concept. The SBE was taken from a palm oil cleaning process. The recycling concept is based on the application of lipolytic clostridia strains. Due to considerably long fermentation times, co‐fermentation with Candida rugosa and enzymatic hydrolyses of the bound oil with a subsequent clostridia fermentation are shown as alternative routes. Anaerobic fermentations under comparison of different clostridia strains were performed with glycerol media, enzymatically hydrolyzed palm oil and SBE. Solutes, side product compositions and productivities were quantified via HPLC. A successful production of ABE solutes from SBE has been done with a yield of 0.15 g butanol per gram of bound glycerol. Thus, the biotechnological recycling of the waste stream is possible in principle. Inhibition of the substrate suspension has been observed. A chromatographic ion‐exchange of substrates increased the biomass concentration.

Adsorptive Pretreatment of Crude Glycerol Prior to Fermentation to 1,3‐Propanediole by Clostridium Diolis

AbstractBei der Gewinnung von Fettsäuren aus Pflanzenölen, z. B. zur Herstellung von Biopolymeren, oder bei der Biodiesel‐ und Seifenproduktion, fällt Glycerin als Nebenprodukt an. Bei der Biokonversion dieses Rohstoffes zu 1,3‐Propandiol wird der Produktionsorganismus Clostridium diolis durch Verunreinigungen im Rohglycerin gehemmt. Als inhibierende Substanzen konnten freie Fettsäuren identifiziert werden. Mithilfe eines adsorptiven Aufarbeitungsverfahrens ist es gelungen, die Fettsäuren zu entfernen und die Konversionseffizienz zu 1,3‐Propandiol zu erhöhen.

STUDY OF ECOLOGICAL INHIBITION OF SULFATE-REDUCING BACTERIAL ACTIVITY AND ASSOCIATED POPULATION SUCCESSION IN OILFIELD FLOODING WATER

In the conventional water flooding process of Daqing Oilfield in China, the occurrence of abundant sulfate-reducing bacterial populations has substantially affected the industrial production. This study was conducted from the perspective of ecological suppression by operating anaerobic ABR reactors and dosing ecological inhibitors in a continuous-flow experiment. The practical operating efficacy was investigated and associated dynamic bacterial population succession was analyzed. Results showed that the sulfate removal rate decreased upon the addition of eco-inhibitor, leading to a low concentration of sulfide products (<0.3 mg/L). Associated redox potential substantially varied from 300 mV to approximately 50 mV, whereas the pH and alkalinity slightly changed and imposed a minor effect on the reaction system. There were significant changes in microbial diversity before and after the eco-suppression, and dominant functional bacterial populations shifted from sulfate-reducing bacteria (Thauera selenatis, Desulfovibrio sp., Clostridium diolis Hydrogenophaga taeniospiralis) to those capable of denitrification (Uncultured firmicute, Clostridium butyricum, Paenibacillus lautus, Arcobacter cibarius). These indicated that the utilization of eco-inhibitor has successfully inhibited the sulfate-reducing bacterial activities.

Mathematical model-based fed-batch cultivation for concentration and productivity enhancement of 1,3-propanediol using Clostridium diolis

Mathematical model-based fed-batch cultivation for concentration and productivity enhancement of 1,3-propanediol using Clostridium diolis

Simple Strategy of Repeated Batch Cultivation for Enhanced Production of 1,3-Propanediol Using Clostridium diolis

Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20% (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8g/L with a productivity of 1.04g/L/h at the end of 65h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.

On-line Characterization of Metabolic State in Batch Cultivation of Clostridium diolis for 1,3-Propanediol Production Using NADH+H+ Fluorescence

NADH is a coenzyme which plays a central role in cellular growth and metabolism. It is an intracellular fluorophore which fluoresces at 460nm when cells are irradiated by 340nm wavelength of light. The application of NADH+H(+) fluorescence measurement for characterization of biomass and its metabolic activity during batch fermentation of 1,3-propanediol (1,3-PD) using Clostridium diolis was investigated in this study. A linear correlation between net fluorescence and biomass concentration was observed during both the initial and final phases of 1,3-PD fermentation. This could be used as an on-line indicator of biomass concentration inside the bioreactor thereby eliminating the need for sampling and off-line analysis for establishing biomass concentration during these phases. Also a sharp decline in the NADH+H(+) fluorescence value was obtained towards the end of fermentation which could be a significant on-line, in situ signal of substrate depletion in the bioreactor and therefore possible fresh nutrient feed for enhanced production of 1,3-PD by repetitive and/or various fed-batch cultivation(s). This is the first report on the use of NADH + H(+) fluorescence measurement technique for 1,3-PD fermentation.

Variations of hydrogen production and microbial community with heavy metals during fermentative hydrogen production

The effects of heavy metals on fermentative hydrogen production were examined based on metal type and concentration. Hydrogen production was stimulated by low concentrations of Cd and Zn but decreased at concentrations of 40 and 1 mg/L, respectively. Hydrogen production was inhibited for the entire range of Cu tested. The order of toxic density was Cu > Zn > Cd at concentrations below 2 mg/L but was Zn > Cu > Cd at higher concentrations. The depression rates of hydrogen production were calculated to be 225.8 mL-H 2/mg-Zn, 67.37 mL-H 2/mg-Cu, and 13.39 mL-H 2/mg-Cd. The presence of heavy metals caused a shift in microbial community. The presence of Clostridium genus bacteria, identified as Clostridium magum, Clostridium diolis, and Clostridium sp., resulted in active hydrogen production. Klebsiella genus bacteria were the most abundant of the class Gammaproteobacteria and also stimulated hydrogen production at relatively low concentrations of heavy metal. When Rhodocista pekingensis, Erwinia chrysanthemi strain 1015-1, Delftia sp. YF 31, or uncultured Klebsiella sp. clone F1 apr.32 were present, hydrogen production was seriously decreased.