Article

Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum.

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Applied and Environmental Microbiology (Impact Factor: 3.95). 06/2007; 73(9):3061-8. DOI: 10.1128/AEM.02296-06
Source: PubMed

ABSTRACT A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA. Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact genes conferring preferential growth under conditions of butanol stress. Since expression using the employed library is possible only from natural promoters, among the enriched genes, we identified 16 genes that constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators (CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respectively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial enrichment approach provided a more detailed understanding of the dynamic process of library enrichment under conditions of selective growth. Further characterization of the genes identified in this study will likely enhance our understanding of the complex phenotype of solvent tolerance.

0 Followers
 · 
74 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The study approach of this paper is to combine experiment and simulation on using butanol as fuel in gasoline engine. First, experiments were performed under full load on a single cylinder spark ignition engine fueled with 35%vol butanol–gasoline blend and the pure gasoline, respectively. The performance and operating parameters were measured. The experimental results showed that with the butanol addition, the ignition timing could be advanced without obvious knocking for higher combustion efficiency. The engine presented superior performance in power, fuel consumption, HC and CO emissions, but deteriorated the NOx emissions largely. Then, a GT-Power simulation model was set-up and calibrated by experimental data. Therefore, the simulation model could be used to study the valve timing impact on engine performance. The simulation results indicated that the enlargement of overlap presented a good “trade-off” effect, such as, decreased emissions (especially for NOx), without deteriorating the torque and fuel consumption too much (except for extremity speeds of 3000 rpm and 8500 rpm, which are not frequently-used in real road driving). The results also indicated that the HC and CO emissions depend more on the fuel properties. The power and NOx emissions depend more on the operating parameters. And the fuel consumption is in between.
    Applied Energy 08/2013; 108:248–260. DOI:10.1016/j.apenergy.2013.03.018 · 5.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO2. Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping.
    08/2012; 2(1):44. DOI:10.1186/2191-0855-2-44
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Analysis of the growth-limiting factor or environmental stressors affecting microbes in situ is of fundamental importance but analytically difficult. Microbes can reduce in situ limiting nutrient concentrations to sub-micromolar levels, and contaminated ecosystems may contain multiple stressors. The patterns of gene or protein expression by microbes in nature can be used to infer growth limitations, because they are regulated in response to environmental conditions. Experimental studies under controlled conditions in the laboratory provide the physiological underpinnings for developing these physiological indicators. Although regulatory networks may differ among specific microbes, there are some broad principles that can be applied, related to limiting nutrient acquisition, resource allocation, and stress responses. As technologies for transcriptomics and proteomics mature, the capacity to apply these approaches to complex microbial communities will accelerate. Global proteomics has the particular advantage that it reflects expressed catalytic activities. Furthermore, the high mass accuracy of some proteomic approaches allows mapping back to specific microbial strains. For example, at the Rifle IFRC field site in Western Colorado, the physiological status of Fe(III)-reducing populations has been tracked over time. Members of a "subsurface clade" within the Geobacter predominated during carbon amendment to the subsurface environment. At the functional level, proteomic identifications produced inferences regarding (i) temporal changes in anabolism and catabolism of acetate, (ii) the onset of N(2) fixation when N became limiting, and (iii) expression of phosphate transporters during periods of intense growth. The application of these approaches in situ can lead to discovery of novel physiological adaptations.
    Frontiers in Microbiology 05/2012; 3:184. DOI:10.3389/fmicb.2012.00184 · 3.94 Impact Factor

Preview

Download
0 Downloads
Available from