Clostridium Cellobioparum Research Articles

Microbial diversity of marine shrimp pond sediment and its variability due to the effect of immobilized media in biohydrogen and biohythane production

Immobilization of microorganism on material is one of the effective methods to improve the production of bio-products including biofuel such as biohydrogen and biohythane (H2 + CH4). In this work, the effect of immobilizing media was studied including clay/activated carbon (CAC), clay/zeolite (CZ) and clay/mixed activated carbon and zeolite (CACZ) on microbial attachment and biogas production including biohydrogen and biohythane using shrimp pond sediment as organic resource and microbial consortium. The result showed that CAC was the most appropriate material for microbial attachment among the other materials. CAC digesters demonstrated higher H2 concentration than control reactors. Interestingly, the control reactors of using only shrimp pond sediment without CAC showed the ability of biohythane production. From 454-pyrosequencing results, it found that Alcaligenaceae family, Tissierella Soehngenia sp. and Planomicrobium sp. were the predominant species in shrimp pond sediment. Meanwhile, the study of microbial species on CAC after digestion process by Illumina MiSeq platform represented Clostridium cellobioparum, Tolumonas auensis and Microbacter margulisiae for bacteria and Methanobacterium aggregans for archaea as predominant species. It was shown that the immobilized media (CAC) favored some types of microbes mainly hydrogen producing microbes and hydrogenotrophic methanogen. This research suggests the CAC as an alternative immobilizing media for bioenergy production especially biohydrogen and biohythane.

Two Peas in a Pod: Sustainable Electrochemical Solutions for Waste and Water Reuse

Satisfying the energy demands of the world population will require the development of technologies for waste and water reuse. Glycerol-containing wastewater is, for example, the major waste product of the biodiesel producers and bioethanol refineries, and it has become an economical and environmental liability to the industry. Purifying, concentrating, and selling the glycerol is no longer a profitable option and producers must pay to dispose of the wastewater as a hazardous waste. Electrochemical platforms show promise for the processing of wastewater and, if driven by customized consortia, they could be used to simultaneously generate added-value products from the organic waste. Hence, we developed an electrochemical reactor for the treatment of glycerin wastewater and the generation of ethanol, which can be used as alcohol feedstock for the transesterification reaction of oils to make the biodiesel industry fully petroleum independent. The electrochemical reactor was driven by the synergistic metabolisms of the exoelectrogen Geobacter sulfurreducens and the bacterium Clostridium cellobioparum, which fermented glycerol into ethanol at high yields (90%) and produced fermentative byproducts that served as electron donors for Geobacter. The synergistic interactions of the microbial catalysts stimulated glycerol consumption and ethanol production. At the same time, the fermentation byproducts were converted into cathodic H2, which facilitated the stripping of ethanol and resulted in clean waste streams that could be reused in upstream reactions. The platform was further improved to process glycerol loadings typical of biodiesel wastewater and to improve ethanol productivity. Furthermore, other alcohols could be produced to improve the economics of the electrochemical platform and address the needs of the producers in a fluctuating market. The results highlight the potential of electrochemically systems to simultaneously treat wastewater and harness the energy in the waste to generate bio-based feedstocks, fuels, and/or specialty or commodity chemicals.

Fermentation of glycerol into ethanol in a microbial electrolysis cell driven by a customized consortium.

The in situ generation of ethanol from glycerol-containing wastewater shows promise to improve the economics of the biodiesel industry. Consequently, we developed a microbial electrolysis cell (MEC) driven by the synergistic metabolisms of the exoelectrogen Geobacter sulfurreducens and the bacterium Clostridium cellobioparum, which fermented glycerol into ethanol in high yields (90%) and produced fermentative byproducts that served as electron donors for G. sulfurreducens. Syntrophic cooperation stimulated glycerol consumption, ethanol production, and the conversion of fermentation byproducts into cathodic H2 in the MEC. The platform was further improved by adaptively evolving glycerol-tolerant strains with robust growth at glycerol loadings typical of biodiesel wastewater and by increasing the buffering capacity of the anode medium. This resulted in additional increases in glycerol consumption (up to 50 g/L) and ethanol production (up to 10 g/L) at rates that greatly exceeded the capacity of the anode biofilms to concomitantly remove the fermentation byproducts. As a result, 1,3-propanediol was generated as a metabolic sink for electrons not converted into electricity syntrophically. The results highlight the potential of consortia to process glycerol in MECs and provide insights into genetic engineering and system design approaches that can be implemented to further improve MEC performance to satisfy industrial needs.

Cellulase Production Potentials of the Microbial Profile of Some Sugarcane Bagasse Dumping Sites in Ilorin, Nigeria

This research work investigated cellulase production potentials of the microbial profile of three sugarcane bagasse dumping sites at Zango area, Ilorin, Nigeria. The microbial isolates were screened for cellulase production with a view to select the best organism for eventual cellulase production. Pour Plate method was used for the isolation and a total of thirteen (13) different organisms including both fungal and bacterial species were isolated and screened. Six (6) fungal isolates identified as Mucor racemosus, Aspergillus niger, Aspergillus flavus, Neurospora sitophilus, Penicillium oxalicum and Penicillium citrinum were isolated, while seven (7) different bacterial species isolated include Clostridium cellobioparum, Clostridium thermocellum,Bacillus subtilis, Bacillus pumillus, Lactobacillus spp, Pseudomonas flavescens and Serratia spp. Generally, bacterial isolates were more in abundance than fungal species. However; fungal isolates were constant and were isolated through the experimental period of three weeks. All the isolates showed cellulase production potential in varying degrees as reflected in the clearance zone around their colonies. Fungal isolates produced more cellulase than the bacterial isolates. Mucor racemosus had the highest clearance zone (75.0 mm) among the fungal isolates while Clostridium cellobioparum (35.0 mm) were the best producer among bacterial isolates. The least producer among fungal isolates, Penicillium citrinum (40.0 mm), is a little more than the bacterial cellulase producer (35.0 mm) and is far greater than the least bacterium Serratia spp (14.0 mm).

Cultural Conditions Necessary for Optimal Cellulase Yield by Cellulolytic Bacterial Organisms as They Relate to Residual Sugars Released in Broth Medium

A total of 115 samples made up of 42 (36.5%) rumen fluid, 36 (31.3%) cowdung and 37 (32.2%) soil samples were collected with the aid of sterile swab sticks except for rumen fluid samples which were collected by use of stomach tubes inserted into mouths of cows and by suction, liquor was collected into prewarmed thermo flasks under continuous flushing with carbondioxide. Soil samples were collected into sterile universal containers. All samples were obtained from abattoirs situated at three locations in Benin City, Nigeria. Samples were investigated for cellulolytic bacteria by Filter Paper Yeast Mineral broth method. Cellulase production was assayed by Carboxymethyl cellulose submerged broth culture while residual sugar yield and other cellulolytic activities were determined by 3, 5 – Dinitrosalicylic acid, Filter Paper, Microcrystalline and Viscometric methods. Cellullolytic bacterial organisms isolated from both soil and rumen fluids were Bacillus subtilis, Clostridium cellobioparum and Clostridium thermocellum. Pseudomonas aeruginosa was isolated from both soil and cowdung samples while Erwinia spp was obtained from both rumen fluid and cowdung samples. Bacillus circulans and Serratia spp were obtained from soil samples only. Clostridium thermocellum and Erwinia spp produced the highest and lowest cellulase yields respectively. All isolates at 40oC and pH 6, recorded optimal sugar yields in culture broth of which Clostridium thermocellum recorded the highest. Lowest yields were recorded at 30oC and pH 3 although there was significant difference in individual yields (P < 0.05). Clostridium thermocellum recorded optimal cellulolytic activities at 50oC and pH 6. All isolates attained optimal cellulolytic activities at 32.6 ± 6.2oC and pH 6.29 ± 0.9 with other broth cultural conditions kept constant. Implications of these findings are discussed.

Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas ( Lama pacos) and sheep ( Ovis aries)

Two bacterial 16S rRNA gene clone libraries were constructed from the forestomach of alpacas and sheep fed alfalfa. After the amplification using the universal 16S rRNA gene primers, equal quantities of PCR products from the same species were mixed and used to construct the two libraries. Sequence analysis showed that the 60 clones from alpacas were divided into 27 phylotypes with 25% clones affiliated with Eubacterium sp. F1. The 60 clones from sheep were divided into 21 phylotypes with 7 phylotypes affiliated with Prevotella ruminicola (40% clones). Clones closely related to Clostridium proteoclasticum, Eubacterium sp. F1, Clostridium cellobioparum, Mogibacterium neglectum, Eubacterium ventriosum, Clostridiaceae bacterium WN011, Clostridium coccoides, Clostridium orbiscindens, Eubacterium sp. F1, Cytophaga sp. Dex80-37, Treponema bryantii and Pelotomaculum sp. FP were only found in the forestomach of alpacas, and those to Anaerovorax odorimutans, Treponema zioleckii, Bifidobacterium indicum, Paludibacter propionicigenes, Paraprevotella clara, Eubacterium siraeum, Desulfotomaculum sp. CYP1, Clostridium bolteae, Clostridium termitidis and Clostridiaceae bacterium DJF_LS40 only in the rumen of sheep. Quantitative real-time PCR revealed that the forestomach of alpacas had significantly lower density of bacteria, with bacterial 16S rRNA gene copies (6.89 [Log10 (copies per gram of wet weight)]), than that of sheep (7.71, P < 0.01). The two clone libraries also appeared different in Shannon index (library from alpacas 3.30 and from sheep 3.04). Our results showed that there were apparent differences in the bacterial diversity and abundance in the forestomach between alpacas and sheep.

Process performance evaluation of intermittent–continuous stirred tank reactor for anaerobic hydrogen fermentation with kitchen waste

A 3-L laboratory scale hydrogen fermentor with fill-and-draw operation defined as intermittent–continuous stirred tank reactor (I-CSTR) was established. There were four operational periods included in this study. In run 1 during the acclimation period, corn starch was added as the auxiliary substrate. The feedstocks of the next two periods were corn starch and kitchen mixture, the Taipei kitchen waste and the Kaohsiung kitchen waste, respectively. Compared between these two periods, run 3 fed with Kaohsiung kitchen waste gives a higher hydrogen producing rate of 27 mmol/L/day than run 2. At run 4 that loading rate was increased and hydraulic retention time was decreased. The maximum hydrogen producing rate of 118 mmol/L/day was found. By the detection of terminal restriction fragment length polymorphism (TRFLP) method, some species of Clostridium such as Clostridium stercorarium, Clostridium thermolacticum, Clostridium aldrichii, Clostridium cellobioparum, Clostridium termitidis (cluster III) and Clostridium formicoaceticum (cluster XI) were presented in all the operational periods. Furthermore, the anaerobic batch test for the substrate utilizing efficiency of the sludge taken from the fermentor was also detected. In the test, the maximum hydrogen producing rate of 48 mmol/L/day was detected. The VSS and the total carbohydrate removal were 35% and 66%, respectively. Organic nitrogen had only a little fraction converted to ammonia, but the oil and grease were not degraded.

Isolation and characterization of Clostridium hobsonii comb. nov.

A search for organisms yielding commercial products from bioresources resulted in a group of 37 isolates from a cattle-waste digester. Using strict anaerobic technique, the organisms were cultured by direct isolation and continuous perfusion on straw, ball-milled cellulose and dewaxed cotton as substrates. Gram-negatively staining rods (though showing a Gram-positive wall in electron micrographs) were straight to slightly-curved with tapering ends, 0·25-0·7 × 1·5-3·5 μm, with peritrichous flagellation. Cultures showed extensive degradation of filter paper; 27–42·7% and up to 31% solubilization of straw and cotton respectively. Cellobiose, -triose and -tetrose, but not glucose, resulted from cellulose hydrolysis. Fermentation products were ethanol, acetate, formate, lactate, succinate and, with some strains, propionate, with CO 2 and H 2 as fermentation gases. Though major tests were done on all, isolates U311, U191, T111, U62 and C400 were examined extensively. All the test strains showed strong to moderate fermentation of glucose, cellobiose, aesculin, maltose, fructose, lactose, mannose, melibiose, ribose, starch, xylose, xylan, raffinose and sucrose. The mol% G + C ratio of the DNA for three strains was 39·5–43·9 ( Tm). Characteristics of the organisms isolated overlapped with those of Eubacterium cellulosolvens NCDO 2433, 2430, ATCC 43171; Clostridium cellobioparum ATCC 15832 and Clostridium thermocellum ATCC 27405. In the present investigation E. cellulosolvens strains produced ethanol, spores in sporulation medium ( Lett. Appl. Microbiol., 1, 31, 1985) and a G + C ratio of 42·5 for strain 2433. C. thermocellum grew at 37·C. Under recommendations 3–4, rule 42 of the international code of nomenclature of bacteria, we propose the name Clostridium hobsonii for U311, a species of biotechnological importance, in honor of P. N. Hobson B.Sc., Ph.D., D.Sc., F.I.Biol., F.R.S.C., F.R.S.E.

Fermentation quality of lucerne laboratory silage treated with &lt;i&gt;Clostridium cellobioparum&lt;/i&gt;, formic acid and Lactomix

Fermentation quality of lucerne laboratory silage treated with &lt;i&gt;Clostridium cellobioparum&lt;/i&gt;, formic acid and Lactomix

Cloning and expression of a Clostridium cellobioparum cellulase gene and its excretion from Escherichia coli JM109

A cellulase gene of Clostridium cellobioparum was cloned on vector pUC19 to form a recombinant plasmid pCMC1 and expressed in Escherichia coli. The expressed enzyme showed carboxymethylcellulase activity but could not degrade Avicel. E. coli JM109 carrying pCMC1 excreted a large amount of cellulase into a culture medium. The C. cellobioparum DNA sequence in the pCMC1 directed the cellulase excretion.

Inorganic nitrogen metabolism in two cellulose-degrading clostridia

Inorganic nitrogen metabolism in two cellulose degrading clostridia, the mesophile Clostridium cellobioparum and the thermophile Clostridium thermocellum was investigated. Both strains show acetylene reduction (i.e. possibly nitrogenase activity), contain glutamine synthetase, glutamate dehydrogenase and glutamate-dependent transaminases. C. cellobioparum additionally contains a NADH-dependent glutamate synthase and a NH 4 + -repressible glycine dehydrogenase (NADPH). Remarkably, acetylene reduction in C. thermocellum is not repressed by ammonium, casting doubt whether this activity is due to nitrogenase. The results are compared with the data from other saccharolytic clostridia.

Inhibitory effects of H2 on growth of Clostridium cellobioparum.

Hydrogen inhibits the growth of hydrogen-producing Clostridium cellobioparum, but not of Escherichia coli or Bacteroides ruminicola. The inhibition is reversible. When hydrogen was removed either by palladium black or by gassing out the tube, glucose utilization increased as did optical density and hydrogen production of C. cellobioparum. Removal of the H2 by methanogenic bacteria favors the growth of C. cellobioparum. Grown with Methanobacterium ruminantium in various concentrations of glucose, the Clostridium reaches a higher optical density and produces more H2 and a higher viable cell count. The cell yield is also higher than in pure culture. In mixed culture, C. cellobioparum produces more acetic acid and less lactic acid, ethanol, and butyric acid than in pure culture. The significance of this metabolic shift and hydrogen utilization in methanogenesis is discussed.

The isolation of anaerobic cellulose-decomposing bacteria from soil.

SUMMARY: A strictly anaerobic mesophilic species of cellulose-decomposing bacteria was isolated from soil by a new technique. Cultures were made in screw-capped bottles with media containing finely divided cellulose. The method and apparatus used for filling these bottles with reduced media and nitrogen is described. Flocculation of cellulose particles in agar media was prevented by incorporating a low concentration of sodium carboxymethyl cellulose. Cellulolytic colonies in cellulose agar media were of two types, punctiform and spreading. The isolate derived from a punctiform colony digested cellulose with the formation of formic, acetic and malic acids, carbon dioxide and hydrogen. Essential growth factors were provided by yeast and soil extracts. Surface colonies on yeast peptone cellobiose agar were differentiated into convex entire central zones and thin transparent irregular margins. In deep culture with the same medium the isolate grew either as discrete lenticular colonies or spread rapidly throughout the agar. These growth forms were unstable, the type appearing in any one culture being unpredictable. In many respects the isolate resembles Clostridium cellobioparum Hungate, and is probably a strain of this species. It differs from C. cellobioparum, however, in producing little or no cellobiose but abundant glucose from cellulose in liquid culture.