Spotlight on biodiversity of microbial cell factories for glycerol conversion

Plant oil based industrial oleochemistry leads to a large side stream of crude glycerol, which offers itself as a low price carbon source for microbial chemical production. Compared to sugar, glycerol is more reduced and less microorganisms are able to use it as carbon source. An interesting feature of glycerol conversion is that many organisms cannot use it as carbon source at all, but they readily use it as electron sink under anaerobic conditions. In any case the number of pathways by which glycerol enters the microbial metabolism is quite limited. Having said this, an interesting variety of products of industrial relevance is accumulated by naturally occurring microorganisms which can use glycerol. These chemicals range from fuels and solvents to polymer precursors up to food additives. The limited number of metabolic pathways and the manageable amount of products allow to highlight the importance of tapping the outstanding resource of biodiversity for industrial purposes. The interplay of microbial biodiversity, metabolic engineering and bioprocess engineering is key for economic success in industrial microbiology. In this article we shed light on the biodiversity of naturally glycerol consuming microorganisms and their impact and importance on microbial chemical production.     

Dynamic flux balance analysis for microbial conversion of glycerol into 1,3-propanediol by Klebsiella pneumoniae

Bioprocess and Biosystems Engineering - To investigate the relationship between the yield of 1,3-propanediol (1,3-PD) and the flux variation in metabolic pathways of Klebsiella pneumoniae, an...     

Application of rapid enzyme assay techniques for monitoring of microbial water quality

Rapid enzyme assay techniques based on direct measurement of beta-d-galactosidase (GALase) or beta-d-glucuronidase (GLUase) activity without selective cultivation are used for rapid estimation of the level of coliform bacteria and Escherichia coli in water samples. Reported detection limits using fluorogenic substrates correspond to culturable target bacteria concentrations that can be appropriate within present guidelines for recreational waters. The rapidity, that is detection within one hour, compromises the specificity of the assay; enzyme activity contributions from other than target bacteria need to be considered, particularly at low levels of target bacteria. Enzyme activities are more persistent than the culturability of target bacteria to environmental and disinfection stress, thus water samples may express enzyme activities of both culturable and viable non-culturable cells.     

Development of a flow-cell system with dual fuel-cell electrodes for continuous monitoring of microbial populations

Summary Two electrode pairs in a flow-through dual fuel-cell system were used to determine the electrochemical potential generated by microbial populations. The potential difference between the counting and the reference electrode pairs was reciprocally proportional to the cell density of microorganisms in the medium, and the response time varied from 20 to 40 minutes at 30°C. Presently, the system is suitable for continuous electrochemical determination of microbial cell populations with a slow growth rate.     

Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem

Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeledPrevotella bryantii specific probe PBB14 were used forin situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation ofP. bryantii andButyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system.P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration ofP. bryantii cellsin vivo (less than 100/nL,i.e. 10⁵ per mL).     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Bacterial conversion of glycerol to beta-hydroxypropionaldehyde

beta-hydroxypropionaldehyde (3-HPA) can be oxidized to acrylic acid, an industrially important chemical used in the manufacture of synthetic plastics and other polymers. Of 19 genera and 55 strains tested, 3 Klebsiella and 2 Enterobacter strains produced 3-HPA. The most efficient strain was Klebsiella pneumoniae NRRL B-4011. Under optimum conditions (28 degrees C; 40 g of semicarbazide hydrochloride per liter, 70 g of glycerol per liter; and pH 6.0), 3.1 g of B-4011 cells per liter accumulated 22 g of 3-HPA per liter at a specific rate of 0.83 g/g per h; however, 14.5 g of cells per liter accumulated 46 g of 3-HPA per liter at a specific rate of 0.41 g/g per h.     

Harnessing electrical-to-biochemical conversion for microbial synthesis

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Development of fecal microbial enzyme mix for mutagenicity assay of natural products

Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.     

Bacterial conversion of glycerol to beta-hydroxypropionaldehyde.

beta-hydroxypropionaldehyde (3-HPA) can be oxidized to acrylic acid, an industrially important chemical used in the manufacture of synthetic plastics and other polymers. Of 19 genera and 55 strains tested, 3 Klebsiella and 2 Enterobacter strains produced 3-HPA. The most efficient strain was Klebsiella pneumoniae NRRL B-4011. Under optimum conditions (28 degrees C; 40 g of semicarbazide hydrochloride per liter, 70 g of glycerol per liter; and pH 6.0), 3.1 g of B-4011 cells per liter accumulated 22 g of 3-HPA per liter at a specific rate of 0.83 g/g per h; however, 14.5 g of cells per liter accumulated 46 g of 3-HPA per liter at a specific rate of 0.41 g/g per h.     

Microbial conversion of glycerol to 1,3-propanediol

Abstract: Glycerol produced by cleavage of natural fats can microbially be converted to 1,3-propanediol (PD) by Citrobacter, Klebsiella and Clostridia strains. The fermentation by C. butyricum , product recovery and purification has been investigated in detail up to the 2 m³ scale. Estimation of product costs for a 10,000 t/a plant indicates that the microbial process is obviously more attractive than the chemical route. Presently, 1,3-propanediol has only a low market volume; however, its use for special polycondensates, in particular polyesters, could reduce glycerol surpluses and make plastics a easily biodegradable part of natural cycles.     

Heat flow measurements in aerobic microbial growth processes with a nonisothermal calorimeter operating directly in the fermenter

A nonisothermal flow calorimeter operating directly in the fermenter was used for heat flow measurements of aerobic microbial growth processes with high biomass productivities. The measuring arrangement makes it possible to describe transitional stages of aerobic yeast cell growth by the ratio of heat production to oxygen consumption (oxy-caloric coefficient). The oxy-caloric coefficient was not constant under the described conditions. The results refer to the existence of an additional energy-delivering mechanism in microbial systems with aerobic carbon source utilization. The mechanism can involve polyphosphate bond division coupled to biomass synthesis.     

Immobilization of glycerol dehydrogenase on magnetic silica nanoparticles for conversion of glycerol to value-added 1,3-dihydroxyacetone

Abstract

Glycerol dehydrogenase (GlyDH) which oxidizes glycerol to the value-added chemical, 1,3-dihydroxyacetone, is of interest due to the oversupply of glycerol as a by-product of the biodiesel industry. To exploit the enzymatic oxidation of glycerol industrially, silica coated magnetic Fe₃O₄ nanoparticles were prepared and then activated with an amino-silane reagent for covalent immobilization of GlyDH via a glutaraldehyde linkage. At the optimal glutaraldehyde concentration of 0.05% (v/v), an enzyme loading of up to 57.5 mg/g-nanoparticles was achieved with 81.1% of the original activity retained. Reaction kinetic analysis indicated that the immobilized GlyDH had almost the same Michaelis-Menten constants for both NAD⁺ and glycerol as the free GlyDH did. However, after immobilization the turnover number k_cat of the GlyDH decreased from 164 s^?1 to 113 s^?1, and the reaction was 1.3-fold less sensitive to inhibition by DHA, which could compensate the decrease in k_cat. The immobilized GlyDH was also less sensitive to changes in pH and temperature, and showed a 5.3-fold improvement in thermal stability at 50^°C. Furthermore, excellent reusability was observed such that 10 cycles of re-use only led to 9% loss of enzyme activity.     

合成微生物群落用于木质纤维素生物质转化的研究进展

木质纤维素在自然界中的储量可观,是生物燃料生产的重要来源。联合生物加工(consolidated bioprocessing)指在不添加酶的情况下,将木质纤维素“一步”转化为生物燃料的过程,在能源危机日益严重的今天具有重要的应用价值。合成微生物群落(synthetic microbial consortia)是由两种或多种纯培养微生物(野生菌株或工程菌株)共同培养而形成的菌群,具有复杂性低、稳定性高等优点,通过协调微生物之间的相互作用以及整个生态系统的稳定,从而实现特定的功能。近年来,合成生物学的快速发展有利于开发新的方法和工具用于合成微生物群落的构建及优化,促进其在联合生物加工方面的应用。本文聚焦于木质纤维素的联合生物加工,综述了合成微生物群落在该领域的研究进展。简单介绍了系统生物学为合成微生物群落的设计提供指导,详细介绍了合成微生物群落的设计原则、优化工具和在实际生产中的应用与挑战,为木质纤维素的联合生物加工提供借鉴意义。     

Identification of an enzyme system for daidzein-to-equol conversion in Slackia sp. strain NATTS

An Escherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacterium Slackia sp. strain NATTS was constructed and screened for E. coli strains having daidzein- and dihydrodaidzein (DHD)- metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis of E. coli strains containing an expression vector incorporating one of the orf-1, -2, or -3 genes revealed that (i) the protein encoded by orf-1 was involved in the conversion of cis/trans-tetrahydrodaidzein (cis/trans-THD) to equol, (ii) the protein encoded by orf-2 was involved in the conversion of DHD to cis/trans-THD, and (iii) the protein encoded by orf-3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase of Lactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in the Slackia sp. NATTS strain proceeds by the action of these three enzymes.     

Optimizing aerobic conversion of glycerol to 3-hydroxypropionaldehyde

When cells of Klebsiella pneumoniae NRRL B-199 (ATCC 8724) were grown aerobically on a rich glycerol medium and then suspended in buffer supplemented with semicarbazide and glycerol, aerobic conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) ensued. Depending on conditions, 0.38 to 0.67 g of 3-HPA were formed per gram of glycerol consumed. This means that up to 83.8% of the carbon invested as glycerol could potentially be recovered as the target product, 3-HPA. Production of 3-HPA was sensitive to the age of cells harvested for resuspension and was nonexistent if cells were cultivated on glucose instead of glycerol as the sole carbon source. Compared with 24- and 72-h cells, 48-h cells produced 3-HPA at the highest rate and with the greatest yield. The cell biomass concentration present during the fermentation was never particularly critical to the 3-HPA yield, but initial fermentation rates and 3-HPA accumulation displayed a linear dependence on biomass concentration that faded when biomass exceeded 3 g/liter. Fermentation performance was a function of temperature, and an optimum initial specific 3-HPA productivity occurred at 32 degrees C, although the overall 3-HPA yield increased continuously within the 25 to 37 degrees C range studied. The pH optimum based on fermentation rate was different from that based on overall yield; 8 versus 7, respectively. Initial glycerol concentrations in the 20 to 50 g/liter range optimized initial 3-HPA productivity and yield.     

A flow injection analysis system for fermentation monitoring and control

An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.     

Development of an automated microbial sensor system

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.     

两相体系中微生物法转化异丁香酚生成香草醛的研究

从土壤中筛选获得一株能耐受高浓度异丁香酚并高效转化生成香草醛的纺锤芽孢杆菌Bacillus fusiformis菌株CGMCC1347,研究了微生物细胞在异丁香酚-水两相体系中转化异丁香酚制备香草醛的过程。在异丁香酚体积分数60％,初始pH 4．0,温度37℃,转速180 r/min的条件下,转化72h,湿细胞质量浓度迭60 g/L时,香草醛质量浓度高达46.10 g/L。