Leona Paulova

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.

Lignocellulosic ethanol: Technology design and its impact on process efficiency

This review provides current information on the production of ethanol from lignocellulosic biomass, with the main focus on relationships between process design and efficiency, expressed as ethanol concentration, yield and productivity. In spite of unquestionable advantages of lignocellulosic biomass as a feedstock for ethanol production (availability, price, non-competitiveness with food, waste material), many technological bottlenecks hinder its wide industrial application and competitiveness with 1st generation ethanol production. Among the main technological challenges are the recalcitrant structure of the material, and thus the need for extensive pretreatment (usually physico-chemical followed by enzymatic hydrolysis) to yield fermentable sugars, and a relatively low concentration of monosaccharides in the medium that hinder the achievement of ethanol concentrations comparable with those obtained using 1st generation feedstocks (e.g. corn or molasses). The presence of both pentose and hexose sugars in the fermentation broth, the price of cellulolytic enzymes, and the presence of toxic compounds that can inhibit cellulolytic enzymes and microbial producers of ethanol are major issues. In this review, different process configurations of the main technological steps (enzymatic hydrolysis, fermentation of hexose/and or pentose sugars) are discussed and their efficiencies are compared. The main features, benefits and drawbacks of simultaneous saccharification and fermentation (SSF), simultaneous saccharification and fermentation with delayed inoculation (dSSF), consolidated bioprocesses (CBP) combining production of cellulolytic enzymes, hydrolysis of biomass and fermentation into one step, together with an approach combining utilization of both pentose and hexose sugars are discussed and compared with separate hydrolysis and fermentation (SHF) processes. The impact of individual technological steps on final process efficiency is emphasized and the potential for use of immobilized biocatalysts is considered.

Use of a mixture of glucose and methanol as substrates for the production of recombinant trypsinogen in continuous cultures with Pichia pastoris Mut

Pure methanol, which is required as an inducer of the AOX1 promoter and a carbon/energy source in processes for recombinant protein production by Pichia pastoris, is impracticable and therefore generally undesirable. As an alternative, a procedure using double carbon substrate was examined (11.7g(carbon)l(-1), 60%/40% carbon from glucose/methanol). The effects on methanol metabolism, extracellular formation of porcine trypsinogen, biomass growth and cell viability were analyzed. In contrast to batch cultures, where the glucose and methanol were utilized sequentially, in carbon/energy-limited continuous cultures (operated between dilution rates 0.03 and 0.20h(-1)) the repressive effect of glucose on methanol utilization was eliminated up to 0.15h(-1) (ca. 130% of μ(max) with methanol). With the mixture, the yield of biomass (1.54±0.12) g(CDW)g(carbon)(-1) was found to be 1.4 times larger than the yield with methanol alone. Despite the current widespread view that glucose has a repressive effect on the AOX1 promoter, the product was synthesized over the entire range of dilution rates, with maximum productivities of (0.70±0.12)mgg(CDW)(-1) h(-1) at 0.07h(-1). Thus, glucose was shown to be a feasible partial substitute for methanol in recombinant protein production by P. pastoris Mut(+) strain while enhancing process productivity.

Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum.

Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, which is a typical feature of clostridial acetone-butanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G+ bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G− bacteria does not allow the uptake and therefore G+ bacteria are stained with higher fluorescence intensity than G− bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G+ bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G− bacteria is detected after initiation of solvent production.

Perspectives of Biobutanol Production and Use

Nowadays, with increasing hunger for liquid fuels usable in transportation, alternatives to crude oil derived fuels are being searched very intensively. In addition to bioethanol and ethyl or methyl esters of rapeseed oil that are currently used as bio-components of transportation fuels in Europe, other options are investigated and one of them is biobutanol, which can be, if produced from waste biomass or non-food agricultural products, classified as the biofuel of the second generation. Although its biotechnological production is far more complicated than bioethanol production, its advantages over bioethanol from fuel preparation point of view i.e. higher energy content, lower miscibility with water, lower vapour pressure and lower corrosivity together with an ability of the producer, Clostridium bacteria, to ferment almost all available substrates might outweigh the balance in its favour. The main intention of this chapter is to summarize briefly industrial biobutanol production history, to introduce the problematic of butanol formation by clostridia including short description of various options of fermentation arrangement and most of all to provide with complex fermentation data using little known butanol producers Clostridium pasteurianum NRRL B-592 and Clostridium beijerinckii CCM 6182. A short overview follows concerning the use of biobutanol as a fuel for internal combustion engines with regard to properties of biobutanol and its mixtures with petroleum derived fuels as well as their emission characteristics, which are illustrated based on emission measurement results obtained for three types of passenger cars.

Production of 2nd Generation of Liquid Biofuels

Fluctuations in the price of oil and projections on depletion of accessible oil deposits have led to national and international efforts to enhance the proportion of energy derived from renewable sources (bioenergy) with special emphasis on the transport sector (e.g. according to Directive 2009/28 EC, by 2020, 20% of energy in EU-27 should be met from renewable sources and 10% should be used in transportation). To fulfil the legal requirements, wider exploitation of biofuels made from renewable feedstocks, as a substitute for traditional liq‐ uid fuels, will be inevitable; e.g. the demand for bioethanol in the EU is expected to reach 28.5 billion litres by 2020 [1], while in America 36 billion gallons of ethanol must be pro‐ duced by 2022 [2]. Bioethanol, which has a higher octane level then petrol but only contains 66% of the energy yield of petrol, can be used as blend or burned in its pure form in modi‐ fied spark-ignition engines [2]. This will improve fuel combustion, and will contribute to a reduction in atmospheric carbon monoxide, unburned hydrocarbons, carcinogenic emis‐ sions and reduce emissions of oxides of nitrogen and sulphur, the main cause of acid rain [2]. Butanol-gasoline blends might outcompete ethanol-gasoline ones because they have bet‐ ter phase stability in the presence of water, better low-temperature properties, higher oxida‐ tion stability during long term storage, more favourable distillation characteristics and lower volatility with respect to possible air pollution. Recently performed ECE 83.03 emis‐ sion tests [3] have shown negligible or no adverse effects on air pollution by burning buta‐ nol-gasoline blends (containing up to 30% v/v of butanol) in spark ignition engines of Skoda passenger cars.

Application of flow cytometry to Saccharomyces cerevisiae peculation analysis

This study was focused on the development and the application of a rapid and reliable staining method for the characterisation of Saccharomyces cerevisiae cells. The experiments were carried out during the shaken flask batch cultivation of yeasts on YEPD medium under aerobic conditions at 27 °C. Stained samples were analysed with an epifluorescence microscope or by employing a flow cytometer. Three different fluorescent probes such as propidium iodide (PI), fluorescein diacetate (FDA), and fluorescein isothiocyanate (FITC) were used for staining. PI was used to determine cell viability in a native sample and DNA content in a sample fixed by ethanol. To assess protein distribution in the yeast population the FITC amine-reactive probe was used. Instantaneous cell enzyme activity was measured as the amount of fluorescein liberated from FDA by intracellular esterase activity.

A study of the physiological response of Pichia pastoris growing in a continuous culture to an induced stress situation

During our work, flow cytometry was used for a study of the influence of cultivation conditions on a physiological state, mainly on the viability and vitality of the recombinant Pichia pastoris strain producing (3-galactosidase. The fluorescent dyes propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol were used for the determination of the structural integrity and the membrane potential of Pichia pastoris cells, respectively. First the staining procedures were optimised and adapted for the Pichia pastoris yeast cells and later applied in a lab-scale continuous cultivation. Stained samples were analysed with the flow cytometer PAS III Partec or the epifluorescence microscope Nikon Eclipse E400. The following conditions were found to be optimum for the staining of Pichia pastoris cells: 57 μg/ml propidium iodide in the sample, 5 min incubation time; 2 μg/ml bis-(1,3-di-butylbarburic acid) trimethine oxonol in the sample, 20 min incubation time. The optimised staining methods were employed in a study of stress-induced physiological response to change of substrate from glycerol to methanol in a glycerol steady state growing culture of Pichia pastoris yeast. During the first 5 h of the transitional state an accumulation of methanol in the culture broth was accompanied by a decreasing concentration of biomass and an increasing amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol. After the adaptation phase the amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol reached steady levels of 2% and 5%, respectively.