Some like it cold: VTT’s cold-tolerant yeast strains for wine and cider production

by Frederico Magalhães

Yeast, like all living organisms, have a narrow range of temperature at which their growth is optimal.  Temperatures below this range tend to slow down or even stop metabolic processes, while higher temperatures can cause cell death.

For many fermentation processes, different temperatures significantly impact product quality.

Ale beer is fermented at a relatively warm temperature (18-24°C) while lager fermentations require lower temperatures (5-15°C). Similarly, red wine is generally fermented at a warmer temperature than the white wine. Fermentation at different temperatures is only possible due to the availability of a variety of yeast species/strains that are adapted to specific temperature conditions.

The yeast Saccharomyces cerevisiae is the most commonly used species for baking, wine, cider and ale fermentations, but is sensitive to low temperatures. When there is a requirement for very low temperature fermentation this species is commonly found in hybrid form with a cold-tolerant species. This is the case for lager yeast.

Through hybridization, lager yeast was able to combine the good fermentative behavior of S. cerevisiae and the cold tolerance of the wild yeast S. eubayanus.

In 2015, VTT generated the first new lager brewing yeast strains in 500 years, proving that it is possible to generate hybrids in a targeted way for specific industrial processes. We recently applied the knowledge obtained in that work to create new yeast strains for the production of cider and wine.

The cold-tolerant Saccharomyces eubayanus

Cold fermentations have some advantages for cider and wine making. At such temperatures, the risk of contamination is low, reducing the need to use sulphite as a preservative. In addition, the aroma profile of white wines is generally improved by using lower fermentation temperatures. White wine and cider are already fermented in relatively cold conditions; therefore, the use of a cold tolerant yeast can potentially improve the process.

The wild yeast S. eubayanus has proven to be one of the most cold-tolerant yeasts known and is partly responsible for the success of the lager beer. In addition, previous results suggest that it contributes to a more diverse aromatic profile and it has good ability for fructose transport. Fructose represents 2/3 of the fermentable sugars in apple juice and half of the sugars in grape must. Due to the total sugar concentration being considerably higher in grape must, incomplete fructose utilization often becomes a limiting step. Therefore, the cold tolerant S. eubayanus has several phenotypic traits relevant for cider and wine making. However, the use of this species is so far limited to lager beer (in hybrid form) and to the spontaneous fermentation of Mudai (a traditional Patagonian beverage prepared with the seeds of the monkey puzzle tree).

New hybrid strains tolerate a wide range of temperatures

We successfully crossed this cold tolerant yeast with a S. cerevisiae wine strain, and tested the fermentative potential in cider and wine making. One of the most important observations was that the new hybrid yeasts tolerated a wide range of temperatures (10-37°C). The lower range allows the yeast to efficiently ferment at low temperatures, while the higher temperatures allow the yeast to resist the stressful conditions associated with the large-scale production in active dry yeast form.

The wider range of temperatures also means that it can serve multiple purposes. Temperature greatly impacts the aromatic properties of cider and wine, and therefore, the aromatic properties can be modulated by simply altering fermentation temperature.

Significantly more pleasant ciders

To test the new hybrid strains we first fermented apple juice at 10 and 20°C. The hybrid yeast outperformed the parental wine yeast. Particularly at 10°C, the wine strain was seriously inhibited by the low temperature. In addition, the hybrids produced more esters, giving a fruity/floral flavour to the ciders. Importantly, unpleasant volatile compounds produced by the wild parent were also eliminated through hybridization.

The sensorial properties of the ciders were evaluated by a consumer panel at the Functional Food Forum of the University of Turku, confirming that the ciders produced by the hybrids were perceived to be significantly more pleasant than those produced by the cold tolerant parent alone.

Wines with more complex and exotic aromatic profiles

To test the potential of the hybrid yeast for wine making, we fermented Sauvignon Blanc and Maccabeu grape musts at laboratory scale. The fermentation performance was comparable to commercial wine yeast strains but with a stronger, more complex and exotic aromatic profile, as described by a panel of expert tasters.

Furthermore, the best performing hybrid yeast was tested for its capacity to maintain high viability after drying and rehydration. The results were very positive with viabilities comparable or even higher than yeast strains commercialized in dried form.

Improved baking yeasts next

We have demonstrated that hybridization is a very powerful technique to improve yeast performance and phenotypic diversity for applications where targeted genetic modification is not yet accepted.  After successfully applying this technique to beer, wine and cider production, we are now assessing hybrids for their potential use by the baking industry. This is being done with the support of a Key Projects grant from the Academy of Finland.

Cold tolerance is also a critical issue in baking. The advent of frozen dough use in recent years represents a challenge for baking yeast. Yeast lose viability when stored frozen, and the poor heat conductivity of dough means that yeast can be exposed to quite low temperatures during the proofing stage of baking. A yeast strain able to perform well at a wider range of temperatures might aid in making loafs of bread evenly fermented after freezing.

References:

Krogerus, K., Magalhães, F., Vidgren, V. & Gibson, B. (2015) New lager yeast strains generated by interspecific hybridization. Journal of Industrial Microbiology and Biotechnology. http://link.springer.com/article/10.1007/s10295-015-1597-6

Magalhães F, Krogerus K, Vidgren V, Sandell M & Gibson B. (2017) Improved cider fermentation performance and quality with newly generated Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids. Journal of Industrial Microbiology and Biotechnology. https://link.springer.com/article/10.1007/s10295-017-1947-7

Magalhães F, Krogerus K, Castillo S, Ortiz-Julien A, Dequin S & Gibson B. (2017) Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking. FEMS Yeast Research. https://doi.org/10.1093/femsyr/fox049

 

DSC_0057xThe author Frederico Magalhães is a third-year PhD student at VTT’s Bioprocess engineering team. His main research interests include studying of yeasts’ natural diversity to develop new hybrid yeasts with relevant phenotypes for the beverage and bread industry. frederico.magalhaes@vtt.fi

How well is CO2 captured by algae in conditions of extreme pH?

By Marilyn Wiebe

Algae, including here microscopic, single cell organisms, macroscopic “seaweeds” and cyanobacteria or “blue-green algae”, are responsible for at least half of the world’s photosynthesis. During photosynthesis, CO2 is taken into a cell, either as CO2 or as bicarbonate, the carbon is converted into biomass or other products and oxygen is released to the atmosphere. The potential for algal capture of CO2 is generally agreed to be higher than that of land plants and many algae are able to exploit environments which are not suitable for traditional agricultural activities, such as areas with brackish or saline water. Algal biomass can be used as short- (e.g. recycling carbon in biofuels) to medium- (e.g. slow release of carbon from fertiliser) term storage of CO2.

Various test and pilot facilities around the world have been investigating the use of different flue gases for algal cultivation. Earlier work at VTT highlighted the methods of transfer of CO2 from production facilities to algal ponds or culture systems. Flue gas may be directly injected into an algal culture system to provide mixing as well as CO2 to the system or it may be captured as carbonate or bicarbonate using an aqueous or chemical scrubber and provided in liquid. Direct injection lowers the pH of aqueous systems, for which low pH tolerant organisms could be beneficial. Alternatively high pH values are needed to ensure that bicarbonate remains in solution and is not released to the atmosphere. Alkaline tolerant algae are desirable when CO2 is fed as bicarbonate/carbonate. These considerations led to questions of how well CO2 is captured by algae in conditions of extreme pH. VTT together with the Finnish Environment Institute SYKE have been investigating some of the species with potential at both extremes.

Three acid tolerant and three alkali tolerant algae were identified for this work, including brown diatoms, green, motile protists, a non-motile green alga and one cold-tolerant, motile, green alga. Each alga has its own growth characteristics, and they did not all grow equally well. However, it was striking that acid tolerant and alkali tolerant strains were equally able to capture CO2 at low (acid tolerant) or high pH (alkali tolerant) as at neutral pH – i.e. uptake of CO2 will not suffer by using acidic or alkali conditions with appropriate strains.

algae-fermentations

Low pH cultures require addition of CO2 but high pH cultures do not

There are, however, differences in operation of the cultures at low or high pH. At low pH the cultures may become CO2 limited and require addition of CO2 in the gas feed (i.e. direct injection of flue gas would be desirable). The low pH strains captured up to about 40% of CO2 from air and up to about 15% of CO2 when it was fed at 2-3%. Although the proportion of CO2 captured is lower when the air is supplemented with CO2 than when it is not, the total amount of CO2 captured by the algae is higher. These acid tolerant strains primarily take up CO2 and one strain was not able to grow at pH values above 7 at which CO2 becomes available primarily as bicarbonate. The acid tolerant strains generally produced more biomass than the alkaline strains.

The alkali tolerant strains did not require CO2 supplementation to grow well (i.e. no flue gas is needed). The diatoms were able to capture up to 60% of the CO2 from air at both pH 7 and pH 9. Provision of additional CO2 in the gas stream resulted in the formation of alkali salts, rather than additional algal growth, so less CO2 was captured when more was provided in alkaline conditions (i.e. direct injection of flue gas would be deleterious). Even at neutral pH, uptake of CO2 from CO2-enriched air was not as efficient as with the acid-tolerant strains. In terms of capturing CO2 from flue gas, providing the CO2 as bicarbonate and recycling some of the salts to the CO2 scrubber should enable the provision of higher amounts of CO2 to such strains with less precipitate formation. A number of recent publications have been addressing the question of how bicarbonate solutions could be better exploited in the cultivation of alkali-tolerant algae.

Algae may also be used to capture CO2 in cool climates

The cold-tolerant alga captures up to about 25% of the CO2 from air, growing slower than the other algae. Nonetheless, this strain also grows similarly at pH 9 as at pH 7 and demonstrates that cold-tolerant algae can provide an option for CO2 capture in areas with cool climates.

As this study draws to an end we are still considering whether bicarbonate solutions could be used with algae growing at near neutral pH values to increase the proportion of CO2 captured.

marilyn-photoDr. Marilyn Wiebe is a principal research scientist at Bioprocess engineering team, with a background in physiological studies and cultivation of yeast and filamentous fungi. During the past 12 years her research has focused on the use of microorganisms, including algae, for biofuels and the development of biorefinery concepts. She is interested in the use of various cultivation methods (photo-, mixo- and heterotrophic, batch, fed-batch and continuous) to understand algal growth and carbon metabolism. This interest has led to several co-authored publications related to algal growth and productivity. marilyn.wiebe@vtt.fi

 

 

 

New, enabling tools for broad utilization of cell factories in sustainable production of chemicals and fuels

By Jussi Jäntti and Dominik Mojzita

Irrespective of the development of oil prices and our technical abilities to retrieve oil from the ground, it is inevitable that in order to fight climate change there is a need to reduce our dependency on fossil fuels and develop sustainable, environmentally friendly production processes using renewable raw materials.

The world we live in has 119 million identified inorganic and organic chemical substances that are either natural or man-made (www.cas.org). The global chemical industry produces roughly 70,000 different chemicals. Oil consumption in Finland is 5.5 liters/day/Finn of which 1 liter/day/Finn contributes raw material for production of chemicals and lubricants (www.oil.fi). Globally, roughly 2 liters/day/person of oil is consumed (www.iea.org).

One way to reduce our oil dependency  is to harness the diversity of biology, especially in the form of microbes to convert waste streams such as pulp and paper industry wood hydrosylates to valuable compounds in bioreactors. This can be achieved when the vast selection of chemical reactions that microbes carry out naturally is combined with novel, designed biochemical pathways. Such pathways can be engineered in an increasing number of microbes using modern genome engineering technologies.

The modified microbes generated in this way can be considered as microbial cell factories that convert a biological raw material into designed end products such as polymer precursors and fuels.

Novel technologies accelerate the development

Significant advances have taken place in our abilities to generate enzymatic production pathways for more and more complex chemicals in microbes due to development of  novel genome engineering techniques (e.g. CRISPR/Cas9), the wealth of DNA sequence data of life on earth, and improved methods in sequence analysis and mathematical modelling of reaction pathways. The first wave of new technologies has already now significantly reduced the time needed for engineering of an industrial, microbial production host. For example, the genome engineering of an industrial yeast strain can now be done over 10 times faster than before. At the same time, these technologies enable engineering of an increasingly broad spectrum of organisms.

Components of a biological system need to be expressed typically at defined levels in order to obtain optimal functionality. This can be important for example for metabolic pathway engineering, where individual genes encoding a production pathway need to be expressed (and the corresponding enzymes produced) in balanced ratios to ensure optimal metabolic flux towards a desired product. Thus, one of the challenges in microbial production host engineering is how to establish specific levels of expression of multiple genes. Furthermore, for many potentially interesting, but currently little used industrial microbes, there are very limited or non-existing tools and/or methods to accomplish controlled expression of heterologous genes. This prohibits efficient use of these hosts in industrial applications.

Need for predictable, stable expression of target genes

An additional level of complexity for the production host engineering comes from the need to maintain predictable and stable gene expression in diverse cultivation conditions or stages of growth. Often, in currently available gene expression systems specific inducing conditions need to be used to achieve desirable expression of target genes. This leads to specific requirements for growth media composition or downstream processing approaches that ultimately result in increased production costs.

In order to achieve predictable and stable expression of target genes in a production organism it is important that the expression of these genes is minimally affected by the intrinsic regulatory mechanisms of the organism. This can be achieved by the use of non-native components (such as promoters and transcription factors) in the engineered gene expression system.

Modular expression system from VTT

We have established a modular expression system that is independent from externally added compound(s) and that enables tight control over a wide range of gene expression levels in the yeast S. cerevisiae (Rantasalo et al. 2016 http://dx.doi.org/10.1371/journal.pone.0148320). A basal, low level expression of a synthetic transcription factor (sTF) is ensured by a core promoter which expression activity is not affected by growth conditions. The sTF is composed of modular DNA binding and gene transcription activating parts that can be used to drive expression of any target gene at tunable levels simply by varying the number of sTF binding sites within the promoter that regulates the expression of the gene of interest.

stf
Importantly, subsequent work has enabled us to extend the published system with a set of novel core promoters and sTFs that can be used to achieve highly tunable expression of target genes, e.g. for optimized metabolic pathways or hydrolytic enzyme mixtures, in a broad range of industrially relevant microbes from various yeasts to filamentous fungi (patent pending).

These systems provide us with novel tools and abilities to engineer eukaryotic microbes for designed purposes using a few, well defined genetic elements. Thus, this system enables us to expand the selection of production organisms that can be effectively engineered for sustainable production of chemicals, fuels and proteins.

If you want to learn more, contact Senior Research Scientist Dominik Mojzita (dominik.mojzita@vtt.fi) or Research Team Leader Jussi Jäntti (jussi.jantti@vtt.fi).

 

jussiDr. Jussi Jäntti leads the Production Host Engineering research team. His team focuses on developing engineering tools for a broad range of eukaryotic microbes. These tools are used to generate cell factories for the sustainable production of chemicals and fuels. jussi.jantti@vtt.fi

 

 

dominikDr. Dominik Mojzita has a strong background in molecular biology and genetic engineering of yeast and filamentous fungi. He has worked on the identification and characterization of novel genes and metabolic pathways, transcriptional and metabolic regulation, genome-wide and gene-specific expression analysis, the production of organic compounds, single-cell analysis, development of synthetic expression tools and the establishment of synthetic control circuits in S. cerevisiae and A. niger. dominik.mojzita@vtt.fi

Novel regulatory factors can help to improve enzyme production in Trichoderma reesei

By Tiina Pakula

In this blog post we describe how we used a systems biology approach to discover new tools for strain improvement in Trichoderma reesei. As a saprophytic fungus by origin living on plant debris, it produces a large repertoire of enzyme activities degrading lignocellulosic material. Genomic analyses have revealed over 200 genes encoding carbohydrate active enzymes, over 60 of them predicted to encode cellulolytic or hemicellulolytic activities.

Based on literature, T. reesei is the most efficient organism for production of enzymes at industrial scale. Optimisation of culture conditions and systematic strain development has made it possible to produce secreted proteins even as much as over 100 g/l.

The enzyme industry exploits T. reesei widely in enzyme manufacturing for e.g. pulp and paper, food, feed, and textile applications. In the recent years the importance of this production host has increased greatly along with the rising trend of producing fuels and chemicals from lignocellulosic feedstocks in second generation biorefineries. We have long experience in developing T. reesei as a producer of lignocellulose degrading enzymes and recombinant proteins. This production platform is the flagship of VTT Expression Service providing custom recombinant protein production.

Group

VTT’s Protein Production team

Understanding of regulatory mechanisms is the key for the enhancement of enzyme production

Production of extracellular enzymes by the fungus is modulated by a multitude of environmental and physiological conditions, the most important determinant being the carbon source available for the fungus.  Hydrolysis of complex plant-derived material requires a synergistic action of different enzymatic activities, and therefore it is beneficial for the fungus to be able to adjust the enzyme production according to the need and to produce suitable combination of enzymes in the presence of the particular material.

Novel biotechnical applications aim at the utilisation of different types of bio-based raw materials in production of useful compounds, and tailor-made enzyme cocktails are needed for efficient utilisation of the material. Understanding of the regulatory mechanisms in production of the enzymes provides tools to enhance the overall production as well as to engineer enzyme cocktails for specific raw materials.

We have applied transcriptomics analysis to examine expression of genes encoding hydrolytic enzymes in the presence of different carbon sources, ranging from defined oligosaccharides to complex plant-derived raw materials such as bagasse, spruce and wheat straw.  Groups of co-regulated genes encoding hydrolytic enzymes were identified on the different carbon source materials, suggesting a potentially important role for them in utilisation of the material.  Furthermore, the analyses revealed novel candidate regulatory genes encoding transcription factors and other regulatory proteins that had a similar expression patterns with the genes encoding the hydrolytic enzymes (Fig. 1).

Fig 1

Figure 1. Heatmap presentation of expression patterns of selected genes in the presence of different lignocellulosic substrates. Red colour in the heatmap indicates induction of the gene by the specific substrates (shown on the bottom). Genes encoding hydrolytic enzymes are shown by green colour and candidate regulatory genes by red on the right.

A set of 28 such candidate regulatory genes were selected for further studies to reveal their possible role in regulation of enzyme production. These genes were first over-expressed in T. reesei with a constitutive promoter.   Seven of the genes tested were shown to increase cellulase and/or xylanase production when overexpressed in the fungal cells (Fig. 2), and some genes appeared to have a negative effect.

Fig 2

Figure 2. Production of (A) xylanase and (B) cellulase activity by strains overexpressing the candidate regulatory genes as compared to the parental strain (parental level shown by red line).

A part of the genes had an effect on production of one particular enzyme activity whereas others appeared to have an effect on production of a broader set of activities. Further analysis of one of the genes, designated ace3, showed that it is essential for cellulase production by the fungus. Overexpression of ace3 (pMH15 in Figure 2) clearly enhanced cellulase and xylanase production, whereas deletion of the gene abolished cellulase production completely and reduced xylanase production significantly (Figure 3).

Fig 3

Figure 3. Production of cellulase and xylanase activity by strains deleted for ace3 and by the parental strain

The multitude of environmental factors affecting production of hydrolytic enzymes and the very large number of enzymes produced by T. reesei suggest that a complex regulatory and signalling network exists in order to maintain coordinated expression of the genes and production of the enzymes. Several regulator genes affecting the expression of cellulase and hemicellulase genes have been identified by us and others over the years, such as the positively acting xyr1 and ace2, and the negatively acting cre1 and ace1. Our results have brought new insights in the understanding of the regulation of the (hemi)cellulolytic system of T. reesei. Novel regulators have been identified, including essential positively acting regulators that can be utilised in further improvement of the production host organism.

Ongoing work focusing on the interplay of the known regulatory factors will lead to better understanding of the regulatory network involved in cellulase gene regulation. This offers tools for the generation of more efficient and robust, production strains of enzymes.

Tiina Pakula

Dr. Tiina Pakula (Principal Scientist) has worked at VTT Technical Research Centre of Finland Ltd since 1994. Her current research topics are related to protein production in microbial host organisms, especially in the filamentous fungus Trichoderma reesei. Application of systems biology approaches in order to understand the cellular processes and to improve the properties of the organism are the main topics of interest. tiina.pakula@vtt.fi

 

 

Additional information and reading:

Home page of VTT Expression Service

Häkkinen, M., Arvas, M., Oja, M., Aro, N., Penttilä, M., Saloheimo, M., and  Pakula, T. 2012. Re-annotation of the GAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates.Microbial Cell Factories 11, 134.

Häkkinen, M.,  Valkonen, M. J., Westerholm-Parvinen, A., Aro, N., Arvas, M., Vitikainen, M., Penttilä, M., Saloheimo, M. and Pakula, T. M. 2014. Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production.  Biotechnology for Biofuels 7,14.

Häkkinen, M.,  Sivasiddhartan, D., Aro, N., Saloheimo, M. and Pakula, T. M. 2015. The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei. Microbial Cell Factories  14, 63.

 

Bringing Mould to Work: Upgrading Citrus Peel and Sugar Beet Pulp

by Peter Richard

For regular households, mould in the food is hardly considered good news. For us research scientists it’s another story. Researching the mould on your onion can open the way to produce chemicals from agricultural wastes that can replace chemicals made from fossil oil.

115 million tons of citrus fruit and 270 million tons of sugar beet are produced annually worldwide which leaves a lot of citrus peel and sugar beet pulp. These raw materials are rich in pectin but of not much use. Sugar beet pulp is dried and used as animal feed. It has to be dried since it otherwise would rot; however drying is very energy consuming. Citrus peel can also be used as animal feed but is often just dumped which is problematic because of the odour. The pectin from citrus peel is sometimes extracted and used as a gelling agent in food, such as jams or marmalades. For this application only 40 – 50 thousand tons are used, i.e. there is a lot of pectin in the world that is unused.

OnionThe mould Aspergillus niger, the black mould, is a common contaminate of foods. If you find black spots on the onion in your kitchen, this is most likely Aspergillus niger. This mould is however also a useful microorganism that is used to produce citric acid. Since the 1930’s citric acid is produced in industrial scale using Aspergillus niger. It is used in food as an acidifier and especially much in soft drinks. Almost all the commercial citric acid, which is currently about 1.6 million tons per year is produced by this mould.

Aspergillus niger ferments also very efficiently pectin which is a polymer made out of D-galacturonic acid monomers. The mould produces pectinase enzymes that hydrolyse the pectin to D-galacturonic acid (oxidised sugar) which is then used as an energy source for growth. We thought that if we want to make something useful out of pectin or D-galacturonic acid, this is a suitable organism. We succeeded to genetically modify the mould so that it would convert D-galacturonic acid to different products, for example galactaric acid which is also called mucic acid.

With the engineered mould the process to produce galactaric acid is very simple. Galactaric acid is not soluble in water at acidic pH which facilitates the downstream processing. The substrates are all soluble and the mould operates well at acidic conditions.

Currently galactaric acid is produced chemically by oxidising the sugar D-galactose using nitric acid. This process is environmentally challenging. Galactaric acid is currently used in skincare products.  In the past, i.e. in the 1930’s it was also used as a food component in self-rising flour. In Mobile, a city in the south of the USA, D-galactose from a wood processing plant was oxidised chemically to produce about 600 tonnes galactaric acid per year.

Besides these applications galactaric acid can be converted chemically to other compounds like adipic acid or furan dicarboxylic acid, FDCA. Adipic acid is used for Nylon. FDCA can also be polymerised. The polymer has good gas barrier properties and can be used for bottles for carbonated drinks. This could replace the PET bottles that are currently used.

Reference: Mojzita et al.Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate. Appl Environ Microbiol. 2010, 76(1):169-75.

100_0046If you want more information about the fungal pathways and D-galacturonic acid catabolism contact the author Dr. Peter Richard. He works at VTT Technical Research Centre of Finland Ltd. as Principal Scientist in Metabolic Engineering research team. His main interests are metabolic engineering of yeast and mould for the production of fuels and chemicals. peter.richard@vtt.fi

A Fresh Look at Old Beer

by Brian Gibson

Archaeological brewing at VTT

The work of an archaeologist is complicated by the fact that the materials under investigation are invariably imperfect. Metal artefacts rust, wood decomposes, DNA degrades, bones and clay vessels shatter. Time leaves only a suggestion of the original materials’ properties and the archaeologist is left to conclude as much as possible from the limited information available. However difficult, with the right approach one can learn much from ancient materials regardless of the size or condition. In this post I write about two of our recent archeological brewing projects: shipwreck beer and sahti.

Shipwreck beer

Shipwreck bottle C49How to interpret information from compromised samples was an issue faced by a team at VTT after receiving two bottles from the ‘Champagne wreck’ – the remains of a schooner found on the sea bed near the Åland Islands in the Baltic sea. Archaeological analysis dated the wreck to sometime in the 1840s. The bottles appeared to contain beer but sensory analysis was impossible due to extensive bacterial spoilage of the beer. Trained beer tasters described the contents in less than favourable terms: rancid, goaty, sulphuric. With such degraded samples it was difficult to imagine how the beer would have originally tasted or how it could be recreated.

The first challenge of the VTT team, led by Dr John Londesborough, was to determine if the contents were, in fact, beer. This was done through sugar analysis which revealed traces of maltose and maltotriose, which are the main sugars present in beer. Further confirmation came from collaboration with partners at the University of Munich who showed unequivocally that the samples contained hops, one of the main ingredients of beer and one which is not found in other beverages. Having established that the samples were beer, it was necessary to determine the level of sample degradation. In particular, it was crucial to determine the level of seawater contamination. The bottles recovered were sealed with corks, which offered only limited protection against the entry of seawater. By determining the concentration of sodium in the beer it was possible to calculate the level of NaCl (salt) and therefore seawater contamination, which in both bottles was approx. 30%.

Vrak Öl Sample collectionAnalysis in Munich  showed that the hop components were extensively degraded, but the concentrations and kinds of degradation compounds could be used to determine what the original compounds were. The presence of hop iso-α-acids indicated that the wort had been boiled prior to fermentation.  A relatively high level of β-acids showed that the hops were less refined than today’s varieties (β-acids have been bred out of modern hop varieties because of their harsh taste). The ethanol concentration (correcting for seawater-contamination) was a relatively mild 4.5% alcohol by volume (ABV). The fruit and floral flavours characteristic of beer were in the normal range, except that the concentrations of 2-phenylethanol were relatively high. This would have imparted a touch of rose aroma to the beers. The presence of a volatile phenol giving a slight clove aroma was also detected. This suggests that the beer may have been ale, as such flavours are not produced by lager yeast.

Detailed microbiological analysis failed to recover any viable yeast cells from the beer, though microscopy revealed abundant dead cells. Likewise, any yeast DNA that may have been present originally was absent – probably degraded enzymatically by the bacteria that appear to have been much more tolerant of the conditions in the bottles during their long storage.

Despite the many challenges faced by the VTT team that analysed the bottles, recreation of the beer, based on the physical and chemical profiling carried out at VTT (and some creative licence) was possible, and Stallhagen’s 1843 beer can now be purchased in Finnish supermarkets.  More detailed results of the study can be found in the Journal of Agriculture and Food Chemistry:

Londesborough, J., Dresel, M., Gibson, B., Holopainen, U., Mikkelson, A., Seppänen-Laakso, T., Viljanen, K., Virtanen, H., Wilpola, A., Kivilompolo, M., Hoffmann, T. & Wilhelmson, A. (2015) Analysis of beers from an 1840s’ shipwreck. Journal of Agriculture and Food Chemistry, DOI: 10.1021/jf5052943.

Sahti: a part of Finland’s living heritage

In 1938, fishermen off the coast of South Africa hauled up an unusual fish. This find was exceptional not because the fish was unknown to science but rather because the species was known only from the fossil record. It had been believed that this fish, the coelacanth Latimeria chalumnae had become extinct 65 million years previously. The coelacanth is one of a number of species that seem to have resisted the effects of evolution over a huge stretch of time. The gingko tree is another notable example which has survived, essentially in the same form, for 170 million years. Darwin, in the Origin of Species wrote:

“…these anomalous forms may almost be called living fossils; they have endured to the present day, from having inhabited a confined area, and from having thus been exposed to less severe competition.”

Such species are uniquely valuable as they provide a direct link to the past. One does not need to base conclusions on scraps of information from long dead and degraded material but can study the real thing in detail. Such studies provide not just a glimpse into the past but also a clearer understanding of how evolutionary forces have shaped modern species; relatives of the lobe-finned coelacanth eventually became the first land animals.  The concept of the living fossil is not necessarily restricted to biological species but can also be applied to the cultural sphere; many traditions, customs, languages, celebrations, etc. have survived unchanged over long periods and offer a unique glimpse into the past. One can even apply this concept to brewing of beer. One relevant example is Finland’s own sahti beer, which appears to have been brewed in more or less the same way for hundreds of years.

Sahti GlassSahti is one of the few ‘ancient’ beer styles still produced in Europe and differs in many respects to modern beers. The main difference is that hops are not an essential ingredient in sahti. Hops have been used to add bitterness and aroma to beer since around 800 AD. Prior to this, a diverse range of ingredients were utilized to add bitterness and balance to beer flavour. These included anise, heather, cinnamon, clove, coriander, liquorice and, as in the case of sahti, juniper. Sahti’s method of production pre-dates the use of hops and is often described as an ancient beer style for exactly this reason. In the EU, sahti is protected by ‘Traditional Speciality Guaranteed’ label, implying that commercial use of the name is restricted to beer produced according to the traditional, registered production method. But what exactly is sahti and how does it differ from modern beers? These questions motivated scientists at VTT to carry out the first comprehensive physical and chemical study of sahti beer.

By analysing samples collected throughout the traditional sahti brewing region of southern and south-western Finland, the researchers showed that, in every respect, sahti was different to commercially available lager, wheat and porter beers. All 12 sahti beers were strong with average alcohol levels of 8% ABV but also with high levels of residual sugars, in some cases more than 100g per litre. Sahti is typically brewed with baker’s yeast rather than brewer’s yeast and this imparts specific flavour characteristics to the beer. In particular, sahti beers contain a trace of clove aroma, which is normally only found in wheat beers. Like English ales, sahti beers have little or no foam – in this case probably because of the high sugar concentration and the absence of wort boiling, both of which are expected to limit foam stability. Other unique features of sahti are a very low level of bitterness due to the absence of hops and an intense fruit flavour due to esters produced by the yeast during fermentation. In some cases the concentrations of fruit compounds were more than ten times higher than in commercial beers. These compounds give sahti its intense banana aroma for example.

As sahti wort is not boiled and contains little or no hops (a natural antimicrobial agent), sahti beer tends to stay fresh for only a short period. The ephemeral nature of the product has probably contributed to its limited geographic distribution and this may, as Darwin contended for biological species, explain why this beer has not ‘evolved’ in the same way as other European beer styles.

Our study has emphasised the uniqueness of sahti beer compared to modern beers and has introduced the international brewing research community to a very individual style of beer and an important part of Finland’s living heritage. Results appear in an upcoming issue of the Journal of the Institute of Brewing:

Jukka Ekberg, Brian Gibson, Jussi Joensuu, Kristoffer Krogerus, Frederico Magalhães, Atte Mikkelson, Tuulikki Seppänen-Laakso & Arvi Wilpola (2015) Physicochemical characterization of sahti, an ‘ancient’ beer style indigenous to Finland. J Inst Brew. In press.

Brian GibsonDr. Brian Gibson has worked at VTT Technical Research Centre of Finland, Ltd. as Senior Scientist and Project Manager since 2009. Brian is responsible mainly for projects relating to brewing yeast biology, fermentation efficiency and beer quality. Current topics of interest include improvement of yeast performance through hybridization and/or adaptive evolution as well as optimization of process conditions. The underlying mechanisms (genetic, molecular, physiological) that govern yeast performance are a main topic of interest. brian.gibson@vtt.fi

Link to Brian’s publications

 

 

 

CoReCo – New metabolic modelling tool for the production strain development

by Mikko Arvas

 
Metabolic engineering is required to make a microbe to produce a new chemical or to improve the production of an existing product. But how to select the right genes and pathways to be engineered?

 
Stoichiometric metabolic modelling encompasses numerous techniques to make these selections using state-of-the-art computational tools and databases of chemical reactions and compounds. At the heart of stoichiometric metabolic modelling are the metabolic models of organisms. In order to model the metabolism of an organism and hence to select the required genetic modifications a metabolic model for that organism is required.

 
Our task at VTT is to develop products, production strains and production processes for the biotechnology industry using microbial production systems. Our focus is on the production of bulk chemicals (for example polymer precursors or biofuels) and proteins (for example biomass degrading enzymes such as cellulases).

 
In collaboration with Aalto University and University of Helsinki, we have developed a novel tool, CoReCo (Comparative ReConstruction), to reconstruct genome wide metabolic models from genome sequence alone (Figure). Unlike previous tools it takes into account information from related species through a phylogenetic approach and verifies the correctness of reactions by atom-maps.

FigureFigure. CoReCo (Comparative ReConstruction) process. Sequence homology searches (InterProScan, Blast and GTG) are carried out for a set of genomes i.e. a genome of interest and some related genomes. A probabilistic model is built for the presence of each enzyme in the set of species and their ancestors. After that atom mapped, electron and element balanced reactions are taken from a reaction database (for example KEGG) to reconstruct a metabolic network of the reactions that the enzymes can carry out. The end product of the process is a Systems Biology Markup Language – model which contains the stoichiometric matrix required for stoichiometric modelling.

 
We have demonstrated the functionality and usability of the reconstructed models with computational steady-state biomass production experiments (Pitkänen et al. (2014) PLoS Computational Biology, 10(2), e1003465). For example, we show that functional models can be built for species that are very distant from major model organisms such as baker’s yeast and for incomplete genome sequences. After the publication we have carried out extensive development of bacterial and fungal reaction databases and also made algorithmic improvements.

 
With novel long read sequencing techniques such as PacBIO, purchasing a high quality genome for a micro-organism starts to be very cost efficient. For example, a yeast genome costs around 5 – 10 000 €. This opens up efficient genetic modifications techniques and now also, with CoReCo, metabolic modelling for any cultivable micro-organism.

 
Well-established microbial production organisms, such as baker’s yeast, are heavily patented and only represent a tiny fraction of natural variability of metabolism. Therefore, exploration of novel production organisms utilizing CoReCo represents considerable opportunities for the industrial biotechnology sector to create new production strains and IPR.

Mikko photoAsk more information about CoReCo and metabolic modelling from the author Senior Scientist Dr Mikko Arvas. He has background in genetics, but for the last ten years he has concentrated on computational genome analysis of fungi for the needs of industrial biotechnology.