Effect of Operating Conditions and Immobilization on Butanol Enhancement in an Extractive Fermentation Using Non-ionic Surfactant.

The present study was undertaken in order to investigate effect of diverse parameters such as fermentation media, pH, initial concentration of biomass, different surfactant concentrations, and immobilization on increasing butanol and total solvent production. Cheng's fermentation media was successfully tested and perceived to increase final solvents concentration. Controlled pH at 12th and 24th hours had negative effect on butanol enhancement; however, it resulted in more butyric acid production which remained accumulated. Ten percent (v/v) biomass was evaluated to increase final solvents concentration and hence butanol yield compared to 20% and 30% (v/v) of initial biomass concentrations. Effect of surfactant concentration (3-20%) was studied on butanol production. Six percent (v/v) L62 resulted in 49% higher final butanol concentration compared to control. Simultaneous immobilization and fermentation showed higher butanol production (16.8 g/L with 6%) which was attributed to partial immobilization of biomass.

Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina.

We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-13C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze-thaw, homogenization, and sonication methods. The limits of quantification were ≤1 µM with excellent linearity for all of the calibration curves ( R2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-13C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation.

A metabolic-activity-detecting approach to life detection: Restoring a chemostat from stop-feeding using a rapid bioactivity assay.

A mediated glassy carbon electrode covered by a thin-film polyviologen was used in the present study to rapidly detect bioactivity in a mixed-culture chemostat (dominated by Clostridium sp.). With the addition of 1mM hexacyanoferrate and 9mM glucose, the current increasing rate (dI/dt) measured under a poised potential of 500mV (vs. Ag/AgCl) can be defined as the quantity of metabolic activity. In the experiment of restoring the chemostat from stop-feeding, it is suggested that when the dI/dt was >2µAmin-1, the influent pump could be directly turned on to maintain the high dilution rate of 0.5h-1; when the dI/dt was lower than 2µAmin-1, reducing the dilution rate would be needed to avoid cell wash out. Since the soluble mediators and polyviologen film will enhance performances by favorable electron transfer and positively charged surfaces, respectively, we suggest that the method can also be employed to detect the bioactivities in environmental samples.

Carbohydrate Depolymerization by Intricate Cellulosomal Systems.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology has been established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium.

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2g/L of AECC and produce 22.1g/L of solvents (4.25g/L acetone, 11.5g/L butanol and 6.37g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.

Spatiotemporal modeling of microbial metabolism.

BACKGROUND: Microbial systems in which the extracellular environment varies both spatially and temporally are very common in nature and in engineering applications. While the use of genome-scale metabolic reconstructions for steady-state flux balance analysis (FBA) and extensions for dynamic FBA are common, the development of spatiotemporal metabolic models has received little attention. RESULTS: We present a general methodology for spatiotemporal metabolic modeling based on combining genome-scale reconstructions with fundamental transport equations that govern the relevant convective and/or diffusional processes in time and spatially varying environments. Our solution procedure involves spatial discretization of the partial differential equation model followed by numerical integration of the resulting system of ordinary differential equations with embedded linear programs using DFBAlab, a MATLAB code that performs reliable and efficient dynamic FBA simulations. We demonstrate our methodology by solving spatiotemporal metabolic models for two systems of considerable practical interest: (1) a bubble column reactor with the syngas fermenting bacterium Clostridium ljungdahlii; and (2) a chronic wound biofilm with the human pathogen Pseudomonas aeruginosa. Despite the complexity of the discretized models which consist of 900 ODEs/600 LPs and 250 ODEs/250 LPs, respectively, we show that the proposed computational framework allows efficient and robust model solution. CONCLUSIONS: Our study establishes a new paradigm for formulating and solving genome-scale metabolic models with both time and spatial variations and has wide applicability to natural and engineered microbial systems.

Phenolic compounds from red wine and coffee are associated with specific intestinal microorganisms in allergic subjects.

The dietary modulation of gut microbiota, suggested to be involved in allergy processes, has recently attracted much interest. While several studies have addressed the use of fibres to modify intestinal microbial populations, information about other components, such as phenolic compounds, is scarce. The aim of this work was to identify the dietary components able to influence the microbiota in 23 subjects suffering from rhinitis and allergic asthma, and 22 age- and sex-matched controls. The food intake was recorded by means of an annual food frequency questionnaire. Dietary fibre tables were obtained from Marlett et al., and the Phenol-Explorer database was used to assess the phenolic compound intake. The quantification of microbial groups was performed using an Ion Torrent 16S rRNA gene-based analysis. The results showed a direct association between the intake of red wine, a source of stilbenes, and the relative abundance of Bacteroides, and between the intake of coffee, rich in phenolic acids, and the abundance of Clostridium, Lactococcus and Lactobacillus genera. Despite epidemiological analyses not establishing causality, these results support the association between polyphenol-rich beverages and faecal microbiota in allergic patients.

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.

Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell.

Clostridium sporogenes forms highly heat resistant endospores, enabling this bacterium to survive adverse conditions. Subsequently, spores may germinate, giving rise to vegetative cells that multiply and lead to food spoilage. Electron microscopy was used to visualise changes in spore structures during germination, emergence and outgrowth. C. sporogenes spores were surrounded by an exosporium that was oval in shape and typically 3 µm in length. An aperture of 0.3-0.4 µm was observed at one end of the exosporium. The rupture of the spore coats occurs adjacent to the opening in the exosporium. The germinated cell emerges through this hole in the spore coat and then through the pre-existing aperture in the exosporium, before eventually being released, leaving behind a largely intact exosporium with an enlarged aperture (0.7-1.0 µm) and coat shell. The formation of this aperture, its function and its alignment with the spore coat is discussed.

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose, and soluble polysaccharides.

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate-binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non-crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid-swollen cellulose is best fitted by the Langmuir two-site model, suggesting that there are two CjCBM3 binding sites on acid-swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.

Enumeration of clostridia in goat milk using an optimized membrane filtration technique.

A membrane filtration technique developed for counting butyric acid bacteria in cow milk was further developed for analysis of goat milk. Reduction of the sample volume, prolongation of incubation time after addition of proteolytic enzyme and detergent, and a novel step of ultrasonic treatment during incubation allowed filtration of goat milk even in the case of somatic cell counts (SCC) exceeding 10(6)/mL. However, filterability was impaired in milk from goats in late lactation. In total, spore counts were assessed in 329 farm bulk goat milk samples. Membrane filtration technique counts were lower than numbers revealed by the classic most probable number technique. Thus, method-specific thresholds for milk to evaluate the risk of late blowing have to be set. As expected, the spore counts of milk samples from suppliers not feeding silage were significantly lower than the spore counts of milk samples from suppliers using silage feeds. Not only were counts different, the clostridial spore population also varied significantly. By using 16S rRNA gene PCR and gene sequencing, 342 strains from 15 clostridial species were identified. The most common Clostridium species were Clostridium tyrobutyricum (40.4%), Clostridium sporogenes (38.3%), Clostridium bifermentans (7.6%), and Clostridium perfringens (5.3%). The 2 most frequently occurring species C. tyrobutyricum and C. sporogenes accounted for 84.7% of the isolates derived from samples of suppliers feeding silage (n=288). In contrast, in samples from suppliers without silage feeding (n=55), these species were detected in only 45.5% of the isolates.

Influence of cell disruption and elution on cellulase release of Clostridium straminisolvens (CSK1).

Clostridium straminisolvens (CSK1) is a novel cellulolytic bacterium isolated from a cellulose-degrading bacterial community MC1. In this study, the influence of the following cell disruption and elution methods on CSK1cellulase release was investigated: (1) freezing-thawing, (2) ultrasonication, (3) elution, (4) freezing-thawing following elution, (5) ultrasonication following elution, and lastly (6) high-pressure homogenization following elution. The activity of the cellulases CMCase, ß-glucosidase, Avicelase, FPase, and xylanase in crude extracts increased 81.5, 23.8, 87.7, 46.3, and 51.7 %, respectively, with an observed optimal treatment method for each cellulase type. The release of protein from CSK1 cells increased following either cell disruption or elution and was highest at 88.3 % in the homogenization high pressure following elution treatment. A newly observed protein was present following cell elution. The performance of cell elution as determined by real time-PCR indicated that the first time cell elution removed more than 90 % of the CSK1 cells from the substrate. These findings demonstrate that cell disruption and elution are effective methods for inducing cellulase release, and elution is the key step for CSK1. To our knowledge, this study presents the first evidence of optimal treatments for induction of cellulase release of Clostridium straminisolvens. This information will be of great value for use in subsequent efforts to better understand the cellulase characteristics of CSK1 and cellulose degradation mechanisms of the MC1 community.

Direct conversion of sugars and organic acids to biobutanol by non-growing cells of Clostridium spp. incubated in a nitrogen-free medium.

Several Clostridium spp. were incubated in a nitrogen-free medium (non-growth medium) containing only butyric acid as a sole precursor for performing butanol production by non-growing cells. Non-growing cells of Clostridium spp., especially Clostridium beijerinckii TISTR 1461, could convert butyric acid to butanol via their sole solventogenic activity. This activity was further enhanced in the presence of glucose as a co-substrate. In addition to glucose, other monosaccharides (i.e., galactose and xylose) and disaccharides (i.e., maltose, sucrose, and lactose) could also be used as a co-substrate with butyric acid. Among the organic acids tested (i.e., formic, acetic, propionic, and butyric acids), only butyric and acetic acids were converted to butanol. This study has shown that it is possible to use the non-growing cells of Clostridium spp. for direct conversion of sugars and organic acids to biobutanol. With this strategy, C. beijerinckii TISTR 1461 produced 12 g/L butanol from 15 g/L glucose and 10 g/L butyric acid with a high butanol yield of 0.68 C-mol/C-mol and a high butanol ratio of 88 %.

Gene replacement and elimination using λRed- and FLP-based tool to re-direct carbon flux in acetogen biocatalyst during continuous CO2/H2 blend fermentation.

A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO2/H2 blend to 245 mM acetate (p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta-ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase (al-adh). Replacement of pta-ack with al-adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO2/H2 blend continuous fermentation (p < 0.005). At the second step, al-adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth (p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack-pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO2/H2 blend fermentation (p < 0.005) with cell duplication time almost nearing that of the wild strain.

Decreased hydrogen production leads to selective butanol production in co-cultures of Clostridium thermocellum and Clostridium saccharoperbutylacetonicum strain N1-4.

When Clostridium thermocellum and Clostridium saccharoperbutylacetonicum strain N1-4 were co-cultured hydrogen production decreased and butanol was selectively produced with extremely low level of acetone. Since the high butanol production correlates with low hydrogen production, the molecular selection of hydrogenase gene activity is expected to yield strains exhibiting a higher butanol ratio.

Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: Clostridium metabolic diversity, tools for process mapping and continuous fermentation systems.

This review emphasises the fact that studies of acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia cannot be limited to research on the strain Clostridium acetobutylicum ATCC 824. Various 1-butanol producing species of the genus Clostridium, which differ in their patterns of product formation and abilities to ferment particular carbohydrates or glycerol, are described. Special attention is devoted to species and strains that do not produce acetone naturally and to the utilisation of lactose, inulin, glycerol and mixtures of pentose and hexose carbohydrates. Furthermore, process-mapping tools based on different principles, including flow cytometry, DNA microarray analysis, mass spectrometry, Raman microscopy, FT-IR spectroscopy and anisotropy of electrical polarisability, which might facilitate fermentation control and a deeper understanding of ABE fermentation, are introduced. At present, the methods with the greatest potential are flow cytometry and transcriptome analysis. Flow cytometry can be used to visualise and capture cells within clostridial populations as they progress through the normal cell cycle, in which symmetric and asymmetric cell division phases alternate. Cell viability of a population of Clostridium pasteurianum NRRL B-598 was determined by flow cytometry. Transcriptome analysis has been used in various studies including the detection of genes expressed in solventogenic phase, at sporulation, in the stress response, to compare expression patterns of different strains or parent and mutant strains, for studies of catabolite repression, and for the detection of genes involved in the transport and metabolism of 11 different carbohydrates. Interestingly, the results of transcriptome analysis also challenge our earlier understanding of the role of the Spo0A regulator in initiation of solventogenesis in C. acetobutylicum ATCC 824. Lastly, the review describes other significant recent discoveries, including the deleterious effects of intracellular formic acid accumulation in C. acetobutylicum DSM 1731 cells on the metabolic switch from acidogenesis to solventogenesis and the development of a high-cell density continuous system using Clostridium saccharoperbutylacetonicum N1-4, in which 1-butanol productivity of 7.99 g/L/h was reached.

Improvement of hydrogen productivity by introduction of NADH regeneration pathway in Clostridium paraputrificum.

To improve the hydrogen productivity and examine the hydrogen evolution mechanism of Clostridium paraputrificum, roles of formate in hydrogen evolution and effects of introducing formate-originated NADH regeneration were explored. The formate-decomposing pathway for hydrogen production was verified to exist in C. paraputrificum. Then NAD(+)-dependent formate dehydrogenase FDH1 gene (fdh1) from Candida boidinii was overexpressed, which regenerate more NADH from formate to form hydrogen by NADH-mediated pathway. With fdh1 overexpression, the hydrogen yield via NADH-involving pathway increased by at least 59 % compared with the control. Accompanied by the change of hydrogen metabolism, the whole cellular metabolism was redistributed greatly.

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 15 g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8 g/L with a productivity of 1.04 g/L/h at the end of 65 h. 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.

Development of high-speed and highly efficient butanol production systems from butyric acid with high density of living cells of Clostridium saccharoperbutylacetonicum.

Living cells are alive and have the butanol-producing ability but not much proliferation under nitrogen source-limited condition. We investigated various butanol production systems with high density of living cells of Clostridium saccharoperbutylacetonicum N1-4 supplemented with methyl viologen (MV) as an electron carrier and nutrient dosing for activity regeneration. In continuous butanol production with high density of living cells, butanol yield was drastically increased from 0.365 C-mol/C-mol with growing cells to 0.528 C-mol/C-mol at a dilution rate of 0.85 h⁻¹, being increased with the butanol to total solvent ratio. This yield was increased to 0.591 C-mol/C-mol by adding 0.01 mM MV. MV addition increased not only butanol yield but also butanol concentration and productivity as compared to those without MV addition. However, living cells lost their activity with incubation time, which lowered the operational stability of the system. Therefore, to maintain constant stability, activity regeneration was carried out with high density of living cells and MV. This system produced butanol at high concentration (9.40 g l⁻¹) and productivity (7.99 g l⁻¹ h⁻¹) for approximately 100 h with maintenance of considerably high yield of butanol (0.686 C-mol/C-mol). Thus, we established a high-speed and highly efficient butanol production system.

Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.