Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.

Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica.

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F ). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L-1  Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L-1  in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L-1  of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.

Production of moth sex pheromones for pest control by yeast fermentation.

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations. Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits. Here we demonstrate a biotechnological method for the production of (Z)-hexadec-11-en-1-ol and (Z)-tetradec-9-en-1-ol, using engineered yeast cell factories. These unsaturated fatty alcohols are pheromone components or the immediate precursors of pheromone components of several economically important moth pests. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting yeast-derived pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a conventionally produced synthetic pheromone mixture. We further demonstrated the production of (Z)-tetradec-9-en-1-yl acetate, the main pheromone component of the fall armyworm Spodoptera frugiperda. Taken together our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control via yeast fermentation.

Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures.

Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose-lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.

Ultrasound-enhanced attenuated total reflection mid-infrared spectroscopy in-line probe: acquisition of cell spectra in a bioreactor.

This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency f(p)) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of f(p) and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

Generation of PHB from Spent Sulfite Liquor Using Halophilic Microorganisms.

Halophilic microorganisms thrive at elevated concentrations of sodium chloride up to saturation and are capable of growing on a wide variety of carbon sources like various organic acids, hexose and also pentose sugars. Hence, the biotechnological application of these microorganisms can cover many aspects, such as the treatment of hypersaline waste streams of different origin. Due to the fact that the high osmotic pressure of hypersaline environments reduces the risk of contamination, the capacity for cost-effective non-sterile cultivation can make extreme halophilic microorganisms potentially valuable organisms for biotechnological applications. In this contribution, the stepwise use of screening approaches, employing design of experiment (DoE) on model media and subsequently using industrial waste as substrate have been implemented to investigate the applicability of halophiles to generate PHB from the industrial waste stream spent sulfite liquor (SSL). The production of PHB on model media as well as dilutions of industrial substrate in a complex medium has been screened for by fluorescence microscopy using Nile Blue staining. Screening was used to investigate the ability of halophilic microorganisms to withstand the inhibiting substances of the waste stream without negatively affecting PHB production. It could be shown that neither single inhibiting substances nor a mixture thereof inhibited growth in the investigated range, hence, leaving the question on the inhibiting mechanisms open. However, it could be demonstrated that some haloarchaea and halophilic bacteria are able to produce PHB when cultivated on 3.3% w/w dry matter spent sulfite liquor, whereas H. halophila was even able to thrive on 6.6% w/w dry matter spent sulfite liquor and still produce PHB.

Identification of lipophilic bioproduct portfolio from bioreactor samples of extreme halophilic archaea with HPLC-MS/MS.

Extreme halophilic archaea are a yet unexploited source of natural carotenoids. At elevated salinities, however, material corrosivity issues occur and the performance of analytical methods is strongly affected. The goal of this study was to develop a method for identification and downstream processing of potentially valuable bioproducts produced by archaea. To circumvent extreme salinities during analysis, a direct sample preparation method was established to selectively extract both the polar and the nonpolar lipid contents of extreme halophiles with hexane, acetone and the mixture of MeOH/MTBE/water, respectively. Halogenated solvents, as used in conventional extraction methods, were omitted because of environmental considerations and potential process scale-up. The HPLC-MS/MS method using atmospheric pressure chemical ionization was developed and tuned with three commercially available C40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C50 carotenoid bacterioruberin, and vitamin MK-8 were the most valuable products found in bioreactor samples. In contrast to literature on shake flask cultivations, no anhydrous analogues of bacterioruberin, as by-products of the carotenoid biosynthesis, were detected in bioreactor samples. This study demonstrates the importance of sample preparation and the applicability of HPLC-MS/MS methods on real samples from extreme halophilic strains. Furthermore, from a biotechnological point-of-view, this study would like to reveal the relevance of using controlled and defined bioreactor cultivations instead of shake flask cultures in the early stage of potential bioproduct profiling.

Stoichiometric and kinetic analysis of extreme halophilic Archaea on various substrates in a corrosion resistant bioreactor.

Extreme halophilic Archaea are extremophile species which can thrive in hypersaline environments of up to 3-5 M sodium chloride concentration. Although their ecology and physiology are widely identified on the microbiological level, little emphasis has been laid on quantitative bioprocess development with extreme halophiles. The goal of this study was to establish, on the one hand, a methodological basis for quantitative bioprocess analysis of extreme halophilic Archaea with an extreme halophilic strain as an example. Firstly, as a novel usage, a corrosion resistant bioreactor setup for extreme halophiles has been implemented. Then, paying special attention to total bioprocess quantification approaches, an indirect method for biomass quantification using on-line process signals was introduced. Subsequently, robust quantitative data evaluation methods for halophiles could be developed, providing defined and controlled cultivation conditions in the bioreactor and therefore obtaining suitable quality of on-line as well as off-line datasets. On the other hand, new physiological results of extreme halophiles in bioreactor have also been obtained based on the quantitative methodological tools. For the first time, quantitative data on stoichiometry and kinetics were collected and evaluated on different carbon sources. The results on various substrates were interpreted, with proposed metabolic mechanisms, by linking to the reported primary carbon metabolism of extreme halophilic Archaea. Moreover, results of chemostat cultures demonstrated that extreme halophilic organisms show Monod-kinetics on different sole carbon sources. A diauxic growth pattern was described on a mixture of substrates in batch cultivations. In addition, the methodologies presented here enable one to characterize the utilized strain Haloferax mediterranei (HFX) as a potential new host organism. Thus, this study offers a strong methodological basis as well as a fundamental physiological assessment for bioreactor quantification of extreme halophiles that can serve as primary knowledge for applications of extreme halophiles in biotechnology.

Investigation of the physiological response to oxygen limited process conditions of Pichia pastoris Mut(+) strain using a two-compartment scale-down system.

Inhomogeneities in production-scale bioreactors influence microbial growth and product quality due to insufficient mixing and mass transfer. For this reason, lots of efforts are being made to investigate the effects of gradients that impose stress in large-scale reactors in laboratory scale. We have implemented a scale-down model which allows separating a homogeneous part, a stirred tank reactor (STR), and a plug flow reactor (PFR) which mimics the inhomogeneous regimes of the large-scale fermenters. This scale-down model shows solutions to trigger oxygen limited conditions in the PFR part of the scale-down setup for physiological analysis. The goal of the study was to investigate the scale-up relevant physiological responses of Pichia pastoris strain to oxygen limited process conditions in the above mentioned two-compartment bioreactor setup. Experimental results with non-induced cultures show that the specific growth rate significantly decreased with increasing the exposure time to oxygen limitation. In parallel more by-products were produced. Examining physiological scalable key parameters, multivariate data analyses solely using on-line data revealed that different exposures to the oxygen limitation significantly affected the culture performance. This work with the small scale-downs setup reflects new approaches for a valuable process development tool for accelerating strain characterization or for verifying CFD simulations of large-scale bioreactors. As a novel methodological achievement, the combination of the two-compartment scale-down system with the proposed multivariate techniques of solely using on-line data is a valuable tool for recognition of stress effects on the culture performance for physiological bioprocess scale-up issues.

Preparation of terbutylazine imprinted polymer microspheres using viscous polymerization solvents.

Spherical micron sized terbutylazine imprinted polymers have been obtained by copolymerization of acrylates in solvent mixtures containing a highly viscous ('oil') component. The new method requires much less organic solvent than precipitation polymerization to produce spherical MIP particles. Different proportions of oil have been used to clarify its role on the morphology. The particles obtained with the optimal composition could be easily packed into short HPLC columns. Chromatographic retention of the template and other compounds has been determined on the novel ('OMIP') columns and was compared to MIP prepared by bulk polymerization. In 70% aqueous acetonitrile eluent the OMIP shows a higher imprinting factor (i. e. less non-selective binding) and lower retention than the bulk MIP. The chromatographic selectivity against template analogs is similar on OMIP and bulk MIP, while the selectivity against non-related substances is better on the OMIP. The effect of oil on the polymer structure appears to be due to its high viscosity.