High-efficiency acetone-butanol-ethanol production and recovery in non-strict anaerobic gas-stripping fed-batch fermentation

Conventional acetone-butanol-ethanol (ABE) fermentation coupled with gas stripping is conducted under strict anaerobic conditions. In this work, a fed-batch ABE fermentation integrated with gas stripping (FAFIGS) system using a non-strict anaerobic butanol-producing symbiotic system, TSH06, was investigated for the efficient production of butanol. To save energy and keep a high gas-stripping efficiency, the integrated fermentation was conducted by adjusting the butanol recovery rate. The gas-stripping efficiency increased when the butanol concentration increased from 6 to 12 g/L. However, in consideration of the butanol toxicity to TSH06, 8 g/L butanol was the optimal concentration for this FAFIGS process. A model for describing the relationship between the butanol recovery rate and the gas flow rate was developed, and the model was subsequently applied to adjust the butanol recovery rate during the FAFIGS process. In the integrated system under non-strict anaerobic condition, relatively stable butanol concentrations of 7 to 9 g/L were achieved by controlling the gas flow rate which varied between 1.6 and 3.5 vvm based on the changing butanol productivity. 185.65 g/L of butanol (267.15 g/L of ABE) was produced in 288 h with a butanol recovery ratio of 97.36%. The overall yield and productivity of butanol were 0.23 g/g and 0.64 g/L/h, respectively. This study demonstrated the feasibility of using FAFIGS under non-strict anaerobic conditions with TSH06. This work is helpful in characterizing the butanol anabolism performance of TSH06 and provides a simple and efficient scheme for butanol production.

     

Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH06

Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to Clostridium acetobutylicum and Bacillus cereus, respectively. They were isolated individually and named as C. acetobutylicum TSH1 and B. cereus TSH2. C. acetobutylicum TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic B. cereus TSH2 did not possess the ability of butanol production; however, it offered C. acetobutylicum TSH1 the viability under non-anaerobic condition. Moreover, B. cereus TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of C. acetobutylicum TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.     

高抗逆高丁比拜氏梭菌的选育及其性能考察简

以抗逆突变株Clostridium beijerinckii IB4为出发菌株,通过常压室温等离子体诱变（ ARTP ）,刃天青平板初筛,摇瓶发酵复筛,筛选出1株高抗逆高丁比的突变菌株C.beijerinckii IT111。发酵结果表明：该突变菌株利用多种C源时均展现其高丁醇比的特性,以玉米芯酸解糖液为C源时,溶剂产量达到10.5 g/L,丁醇8.0 g/L,丁醇比高达76％。抑制物抗逆性测试结果显示：糠醛和酸类对C.beijerinckii发酵影响较小,酚类物质对C.beijerinckii抑制作用较强,其中以香草醛为最。综上所述,C.beijerinckii IT111是1株极具潜力的利用木质纤维原料制备丁醇的菌株。     

Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus.

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.     

New options to engineer biofuel microbes: Development and application of a high-throughput screening system

The number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium acetobutylicum are quite limited, demonstrating the physiological complexity of solventogenic clostridia. Since multiple largely unknown parameters determine a particular phenotype, an inverse strategy to select a phenotype of interest can be useful. However, the major constraint for explorative or combinatorial metabolic engineering approaches is the availability of a feasible screening method to select the desired phenotype from a large population in a high-throughput manner. Therefore, a semi-quantitative assay was developed to monitor alcohol production in microtiter cultures of C. acetobutylicum. The applicability of the screening system was evaluated by two examples. First, C. acetobutylicum ATCC 824 was chemically mutagenized and subjected to high butanol concentrations as a pre-selection step. Screening of the butanol-tolerant population resulted in the identification of mutants with >20% increased butanol production as compared to the wildtype. The second application example was based on a pre-engineered C. acetobutylicum strain with low acetone biosynthetic activity, but concomitantly reduced butanol titer. After chemical mutagenesis, a total of 4390 clones was analyzed and mutants with significantly increased butanol concentrations and similarly low acetone levels as the parental strain were selected. Thus, the suitability of the semi-quantitative screening system was validated, opening up new perspectives for combinatorial strategies to improve solventogenic clostridia and other biofuel microbes.     

Analysis of 16S–23S rRNA gene internal transcribed spacer of Vibrio anguillarum and Vibrio ordalii strains isolated from fish

The basis of the bactericidal action of antibiotics and the mechanisms of antibiotic tolerance are largely unknown. To elucidate one of the mechanisms of antibiotic tolerance, the present study investigated the role of Pseudomonas aeruginosa quorum sensing (QS) and the rpoS gene in antibiotic tolerance. The survival rates of the lasR and lasI mutants were observed to be lower than that of the parental strain in time-dependent killing studies with 8 μg mL⁻¹ ofloxacin, but the survival rates of the rhlR and rhlI mutants were not different from that of the parental strain. Moreover, a lasR -overexpressing strain was more tolerant to ofloxacin than the parental strain, but this was not the case for an rhlR -overexpressing strain. The mRNA expression levels of lasR , lasI , and rpoS in the wild-type strain in the presence of bactericidal concentration of ofloxacin were lower than that in the absence of ofloxacin. In addition, the significant loss of antibiotic tolerance in the lasR mutant was recovered by the overexpression of rpoS . These results suggest that the Las QS system in P. aeruginosa is involved in the development of ofloxacin tolerance, and the tolerance induced by the Las-system is regulated by rpoS gene.     

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

高浓度丁醇浸泡及N＋束注入诱变双重作用筛选丁醇高产菌

通过高浓度丁醇浸泡处理丙酮丁醇梭菌（Clostridiumacetobutylicum）CL-2,筛选得到一株丁醇耐受能力提高并溶剂产量增加的菌株BR30—2,丁醇产量达11．77g/L,比CL-2提高了16．65％。以BR30—2作为出发菌株,进行N＋束注入诱变,筛选得到高产菌株BH．9,丁醇产量达14．5g/L,总溶剂为23．14g／L。在BH-9发酵过程中添加0．1％丁酸钠,丁醇产量达到16．59g/L,丁醇比例提高至67．38％。     

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

Mechanism of Nitrate Tolerance in Tn5 Mutants of Cicer-Rhizobium Strains

The activities of nitrate reductase (NR) and nitrite reductase (NiR) and production of indole-3-acetic acid (IAA) by symblotic nitrate tolerant Tn5 mutant AC-10 of Cicer-Rhizobium strain F-75 and mutants BC-35 and BC-46 of strain G36-84 developed earlier, have been studied under ex planta condition. The rhizobiaI mutants and their parental strains were grown with nitrate (0.0, 0.5, 1, 2 or 4 mM), aerobically and microaerobically. The overall activities of NR were 70–91% lower in aerobically grown and 78–87% lower in microaerobically grown mutant cells compared to their parental strains. Similarly, the overall activities of NiR were 36–55% and 27–37% lower in aerobically and microaerobically grown mutant cells, respectively, compared to their parental strains. On the contrary, the overall production of IAA in the culture medium by aerobically grown mutant cells was significantly higher compared to their parental strains. Based on these results, it has been suggested that impaired NR activity and a favourable NiR/NR ratio preventing nitrite accumulation in the rhizobial mutants, may be responsible for imparting nitrate tolerance to chickpea - Rhizobium symbiotic system.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.     

Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.     

Isolation, characterization and evaluation of hyper 2-propanol producing bacteria from Singapore environment

Three hyper 2-propanol producing strains were isolated from Singapore environment using an enrichment step and a high through-put screening step. The analysis of the amplified 16S rDNA revealed that the isolates belonged to Clostridium species and they were named as Clostridium sp. BT10-1, Clostridium sp. M10-1 and Clostridium sp. PU31-4. At 1 L scale, the 2-propanol titer of these positive strains was 1.6–2.1 times of that of Clostridium beijerinckii NRRL B593, which is so far the most efficient natural 2-propanol producer. The highest 2-propanol titer was achieved by isolate BT10-6 and it was 5.26 g/L (87.5 mM). These three positive strains BT10-6, M10-1 and PU31-4 consumed glucose almost completely in 40–48 h and gave 2-propanol productivity at 0.132, 0.118 and 0.087 g/L/h, respectively, which is 3.0–4.6 times of 0.029 g/L/h given by C. beijerinckii NRRL B593. Butanol was also produced by these positive strains with a slightly lower butanol titer and higher butanol productivity, compared to butanol control strain C. beijerinckii NCIMB 8052.     

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.     

苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。     

Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling

Genome shuffling based on cross mating was used to improve the tolerance of the pentose-fermenting yeast Pichia stipitis towards hardwood spent sulphite liquor (HW SSL). Six UV-induced mutants of P. stipitis were used as the starting strains, and they were subjected to 4 rounds of genome shuffling. After each round, improved strains were selected based on their growth on HW SSL gradient plates. Mutant libraries were established after each round and these improved mutant strains served as the starting pool for the next round of shuffling. Apparent tolerance to HW SSL on the gradient plate increased progressively with each round of shuffling up to 4 rounds. Selected improved mutants were further tested for tolerance to liquid HW SSL. After 4 rounds of shuffling, 4 mutants, two from the third round (designated as GS301 and GS302) and two from the fourth round (designated as GS401 and GS402), were selected that could grow in 80% (v/v) HW SSL. GS301 and GS302 grew also in 85% (v/v) HW SSL. GS301 was viable in 90% (v/v) HW SSL, although no increase in cell number was seen. The P. stipitis wild type strain (WT) could not grow on HW SSL unless it was diluted to 65% (v/v) or lower. Genome-shuffled strains with improved tolerance to HW SSL retained their fermentation ability. Fermentation performance of GS301 and GS302, the 2 strains that exhibited the best tolerance to liquid HW SSL, was assessed in defined media and in HW SSL. Both strains utilized 4% (w/v) of xylose or glucose more efficiently and produced more ethanol than the WT. They also utilized 4% (w/v) of mannose or galactose and produced ethanol to the same extent as the WT. GS301 and GS302 were able to produce low levels of ethanol in undiluted HW SSL.     

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production

Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ～60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.