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 C-40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C-30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C-50 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

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\ufeff 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

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\ua0Lactococcus lactis\ua0using 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\ua0lacSgene, 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

Current state and challenges for dynamic metabolic modeling

While the stoichiometry of metabolism is probably the best studied cellular level, the dynamics in metabolism can still not be well described, predicted and, thus, engineered. Unknowns in the metabolic flux behavior arise from kinetic interactions, especially allosteric control mechanisms. While the stoichiometry of enzymes is preserved in vitro, their activity and kinetic behavior differs from the in vivo situation. Next to this challenge, it is infeasible to test the interaction of each enzyme with each intracellular metabolite in vitro exhaustively. As a consequence, the whole interacting metabolome has to be studied in vivo to identify the relevant enzymes properties. In this review we discuss current approaches for in vivo perturbation experiments, that is, stimulus response experiments using different setups and quantitative analytical approaches, including dynamic carbon tracing. Next to reliable and informative data, advanced modeling approaches and computational tools are required to identify kinetic mechanisms and their parameters.The authors EV, AT, KN, IR, MO, DM and AW are part of the ERA-IB funded consortium DYNAMICS (ERA-IB-14-081, NWO 053.80.724)

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Background Conventional experiments in small scale are often performed in a Black Box fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. Results The same mastermix (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). Conclusions The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities

Experimental Microbial Evolution of Extremophiles

Experimental microbial evolutions (EME) involves studying closely a microbial population after it has been through a large number of generations under controlled conditions (Kussell 2013). Adaptive laboratory evolution (ALE) selects for fitness under experimentally imposed conditions (Bennett and Hughes 2009; Dragosits and Mattanovich 2013). However, experimental evolution studies focusing on the contributions of genetic drift and natural mutation rates to evolution are conducted under non-selective conditions to avoid changes imposed by selection (Hindré et al. 2012). To understand the application of experimental evolutionary methods to extremophiles it is essential to consider the recent growth in this field over the last decade using model non-extremophilic microorganisms. This growth reflects both a greater appreciation of the power of experimental evolution for testing evolutionary hypotheses and, especially recently, the new power of genomic methods for analyzing changes in experimentally evolved lineages. Since many crucial processes are driven by microorganisms in nature, it is essential to understand and appreciate how microbial communities function, particularly with relevance to selection. However, many theories developed to understand microbial ecological patterns focus on the distribution and the structure of diversity within a microbial population comprised of single species (Prosser et al. 2007). Therefore an understanding of the concept of species is needed. A common definition of species using a genetic concept is a group of interbreeding individuals that is isolated from other such groups by barriers of recombination (Prosser et al. 2007). An alternative ecological species concept defines a species as set of individuals that can be considered identical in all relevant ecological traits (Cohan 2001). This is particularly important because of the abundance and deep phylogenetic complexity of microbial communities. Cohan postulated that “bacteria occupy discrete niches and that periodic selection will purge genetic variation within each niche without preventing divergence between the inhabitants of different niches”. The importance of gene exchange mechanisms likely in bacteria and archaea and therefore extremophiles, arises from the fact that their genomes are divided into two distinct parts, the core genome and the accessory genome (Cohan 2001). The core genome consists of genes that are crucial for the functioning of an organism and the accessory genome consists of genes that are capable of adapting to the changing ecosystem through gain and loss of function. Strains that belong to the same species can differ in the composition of accessory genes and therefore their capability to adapt to changing ecosystems (Cohan 2001; Tettelin et al. 2005; Gill et al. 2005). Additional ecological diversity exists in plasmids, transposons and pathogenicity islands as they can be easily shared in a favorable environment but still be absent in the same species found elsewhere (Wertz et al. 2003). This poses a major challenge for studying ALE and community microbial ecology indicating a continued need to develop a fitting theory that connects the fluid nature of microbial communities to their ecology (Wertz et al. 2003; Coleman et al. 2006). Understanding the nature and contribution of different processes that determine the frequencies of genes in any population is the biggest concern in population and evolutionary genetics (Prosser et al. 2007) and it is critical for an understanding of experimental evolution

Dynamic Experiments for Bioprocess Parameter Optimization with Extreme Halophilic Archaea

The to-date studies on extreme halophiles were focused on shake flask cultivations. Bioreactor technology with quantitative approaches can offer a wide variety of biotechnological applications to exploit the special biochemical features of halophiles. Enabling industrial use of Haloferax mediterranei, finding the optima of cultivation parameters is of high interest. In general, process parameter optimizations were mainly carried out with laborious and time-consuming chemostat cultures. This work offers a faster alternative for process parameter optimization by applying temperature ramps and pH shifts on a halophilic continuous bioreactor culture. Although the hydraulic equilibrium in continuous culture is not reached along the ramps, the main effects on the activity from the dynamic studies can still be concluded. The results revealed that the optimal temperature range may be limited at the lower end by the activity of the primary metabolism pathways. At the higher end, the mass transfer of oxygen between the gaseous and the liquid phase can be limiting for microbial growth. pH was also shown to be a key parameter for avoiding overflow metabolism. The obtained experimental data were evaluated by clustering with multivariate data analyses. Showing the feasibility on a halophilic example, the presented dynamic methodology offers a tool for accelerating bioprocess development

Characterization of the respiratory physiology of Lactococcus lactis for starter culture production with improved acidification capacity

Commercial freeze-dried starter cultures for cheese making are produced mainly via anaerobic batch processes. A recent discovery has shown that some lactic acid bacteria (LAB) are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective [1]. Respiration is energetically beneficial: compared to fermentation, under respiratory conditions the biomass yield is higher and a different by-product pattern is observed. However, it is also important to consider whether the different metabolism can affect the performance of the starter culture. Thus, this project investigates LAB respiratory physiology, aiming to clarify the molecular reasons behind the milk acidification capacity of the respiratory culture. Our bioreactor results demonstrate that with hemin addition, cells switch from the fermentation to respiration only in the late exponential phase of growth. Although presence of oxygen is an additional stress for LAB, in the presence of hemin under aerobic conditions cells have surprisingly better fermentation behaviour, i.e. higher lactate yield before the respiratory switch. Therefore, we hypothesize that with improved fermentation a certain energy threshold is achieved for the respiratory switch. This energy requirement might be related to the intake of the hemin, however this aspect needs further investigation, as hemin transport into the cells has not been characterized yet.Reference: [1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):14

What induces respiration in lactic acid bacteria? Characterization of respiratory metabolism of Lactococcus lactis in bioreactors for production of starter cultures with improved acidification capacity

Commercial freeze-dried lactic acid bacteria starter cultures for cheese making are produced mainly via anaerobic batch fermentations. Recently, it has been shown that some Lactococcus lactis species are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective. Respiration is energetically beneficial and under respiratory conditions, higher biomass yield is obtained together with a changed by-product pattern, compared to fermentation [1]. So far it has not been studied how the different culture conditions and thereby different metabolism affect the starter culture performance. In this project, we investigate respiratory culture conditions, and the effect on the milk acidification capacity of the culture. Since Lactococcus lactis is a fastidious microorganism, a rich chemically defined medium was developed to support the nutrient requirements, and was applied for bioreactor cultivations with quantitative approaches. The product profile and on-line gas analysis revealed that with hemin addition at the start of the process, cells switch to respiratory metabolism only in the second phase of growth, after an initial mixed-acid fermentative phase. To characterize the observed respiratory switch, a multivariate study was performed: a set of bioreactor batch experiments were carried out with different initial sugar concentrations under anaerobic, aerobic, and respiratory conditions. The results indicate that hemin addition together with some yet not defined threshold must be met in order to induce the respiratory metabolic state of the culture.[1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):143‐14

Which methods for viable yeast cell quantification can be used in lignocellulosic fermentation processes

Cell concentration is a primary characteristic of fermentation processes. The total cell concentration in aparticle-free liquid medium can be easily assessed by cell counts, optical density or dry weight. The quantification of viable cells is not as straightforward. Viable cells can be defined as culturable, metabolically active and intact cells. Culturable cells can be assessed by colony-forming unit (CFU) assay. Metabolically active and intact cells have been quantified by e.g. qPCR, dielectric spectroscopy probes, and flow cytometry using various dyes. All these methods work well for applications in clear liquid media, but have not been validated in 2nd generation bioprocesses using lignocellulosic materials.In this study we evaluate the applicability of several methods for quantitative assessment of both total and viable cell concentrations in lignocellulosic media. In order to mimic typical conditions of lignocellulosic fermentations, we used a central composite design of experiments with known cell numbers, water insoluble solids content (WIS) and osmolality as factors. For the osmolality, we used sorbitol and NaCl to differentiate hyperosmotic conditions at different ion strengths and conductivities. The cell concentrations were determined using cell enumeration in a hemocytometer (with and without methylene blue staining), plating and enumeration of CFU, qPCR on extracted DNA and RNA, and on-line permittivity using a capacitance probe. These methods have the potential to be less affected by impurities and water insoluble solids in lignocellulosic media than e.g. dry weight and turbidity. The number and viability of cells used to create the test conditions of the experimental design were first determined from the seed culture on defined mineral medium. Considering all experimental points and some validation points within the design space, all the selected methods were used for measuring total and viable number of cells. With these data we built a quantitative model to fit all interaction effects and curvature, and to calibrate the qPCR and permittivity results to the number of total and culturable cell counts. Data of qPCR on DNA were fitted to total cell numbers, WIS level and osmolality. The permittivity measured by the dielectric probe was fitted to CFUs, WIS level, osmolality and measured conductivity. Parameter optimization resulted in statistically significant models with good predictive capacity. The results showed that cell counts and CFU were not sensitive to WIS and osmolarity levels. Therefore they can be used asreference methods in lignocellulose-based media. Furthermore, using the selected methodologies in simultaneous saccharification and fermentation (SSF) process of pre-treated wheat straw showed consistent results in total and viable cell numbers.Development of reliable and validated total and viable cell quantification methods will contribute to wellmonitored lignocellulosic fermentation processes both for research and industry in bio-based production