Today we’re going to go over an article from the journal Food Chemistry that came out in 2006 called “Low temperature brewing using cells immobilized on brewer’s spent grains” by Nikolaos Kopsahelis, Maria Kanellaki and Argyro Bekatorou.
Foreword by Me:
As some of you may have heard, there is a new technology dawning in the brewing world that even started trickling down even to the homebrewing level. I am talking about immobilized yeast. Some of you may have heard of a group giving away magic beads that will ferment your wort without having to deal with suspended yeast for homebrewer feedback. I contacted the group and they seemed happy to give me a sample to try and write a feedback on my blog, but suddenly disappeared and didn’t answer my emails anymore while giving out samples to others. In any case, as far as I know, the results are predictable – quick and clean fermentation, but the end product lacks the complexity of normal ale fermentation. This may seem like awesome new tech, but it actually goes back to the 1970s when scientists first tried to create these for ethanol production and wine making. Brewing industry quickly picked up on that and have been looking into possibility of using this technology to lower proaction costs. The original cell immobilization was done by S. Humphreys’ group in 1976 and they utilized metal hydroxides as the carrying matrix for immobilized cells. In 1992, a group from Greece under A. Koutinas isolated a strain of S. cerevisiae from a grape that was not only alcohol resistant, but also psychrotolerant (grows best at low temperature) and did not create unpleasant off-flavors during fermentation, making it an interesting candidate for wine and beer production. It became known as AXAZ-1 strain. By that time, it became apparent that metal based matrix is not ideal for cell immobilization and scientists started experimenting with organic materials, particularly those rich in cellulose. With recycling becoming more and more popular, beer scientists started looking at best ways to utilize the spent grain. Spent grain leftover from the mashing process is the largest byproduct of beer making process and provides a matrix rich in cellulose, protein and starch. It became quickly apparent that yeast can be easily immobilized on spent grain and the resulting mix was effective in fermenting wort. Most of the research focused on refining and optimizing this process was done in Greece, but with the financial crisis that has mostly ruined Greek economy, I’m afraid the progress has been delayed by at least a few years. The following article looks at fermentation properties of AXAZ-1 immobilized on spent grain. Overall the article is technical and concentrates on analysis aspects, but I’ll try to sum it up in general terms. As before, I claim no authorship of any of these studies and credit goes to the scientists who tirelessly worked on creating this paper.
Article Summary:
Use of immobilized cells has been studies for well over three decades with main goals being facilitation of continuous processing, shortening maturation time and production cost reduction. This process has already been used for commercial production of low alcohol beers as well as secondary fermentation. There are, however, several aspects to this that require improvement in order to become commercially valuable, not the least of which is consumer fears of new technologies. Others include quality of such yeast, preparation costs, handling and regeneration, consistent fermentation characteristics, dealing with contamination and maintenance of these cultures within the brewery.
One way of improving on some of these issues would be optimization of the carrying matrix on which the cells are immobilized. Researchers have used various food grade supports such as gluten pellets, processed cellulotic materials and even fruits, resulting in beers with good taste and aromas even when fermented at very low temperatures of 0-5˚C (32-41˚F). When selecting a good carrying matrix, one also has to consider availability and the ease with which it’s made. With recycling becoming more fashionable every day, researchers turned their sights on the spent grain. As the biggest brewing byproduct, it is something breweries are never short of, making it a very easily accessible material. Moreover, it is a material rich in fiber, proteins and lipids that is also 100% compatible with beer production. Spent grains have already been utilized as protein rich animal feed and mushroom growing substrate. European Social Fund funded efforts to upgrade spent grain usage in beer and alcohol fermentation, serving as a fermentation biocatalyst based on cell immobilization. Combining spent grain and cold-loving yeast, therefore, presented an interesting possibility, which was investigated in this paper.
Methods: I omitted most of the technical stuff that would be of no interest to homebrewers.
– Preparation of the support and immobilization of cells: Spent grains were delignified (removal of lignin which gives plants their rigidity and woodiness) by boiling in 1% solution of NaOH for 3 hours. Delignified Brewer Spent Grains (DBSG) were then washed with water, drained and sterilized by autoclaving. Yeast was immobilized on the grain by suspending AXAZ-1 cells in 12% glucose synthetic medium and DBSG were mixed into that solution. CO2 produced during fermentation was sufficient enough to gently mix the grain. Fermentation was allowed to progress for 6-8 hours until all sugar has been consumed. The remaining liquid was decanted and the grain (with yeast cells now stuck to it) was washed twice with fresh glucose medium and used for brewing experiments.
– Fermentation: Biocatalyst and 1.048 SG wort were mixed in ratio of 1:2 weight:volume and fermented at 15˚C (59˚F) to adapt cells to wort. Following fermentations were carried out with successive temperature reductions to 10, 5 and 0˚C (50, 41 and 32˚F). At the end of each fermentation, the biocatalyst was washed with fresh wort and used to ferment the next batch for a total of 10 consecutive small batches (650mL). Green beers were immediately collected and analyzed for ethanol, residual sugar, free cells, diketones, dimethyl sulfide (DMS), bitterness, color and volatiles. Green beers were tasted by 10 non-trained people in accordance to taste test protocol, as well as by an industry trained taster from the Athenian Brewery.
Results:
– Fermentation: Attachment of yeast cells to DBSG was confirmed by electron microscopy (Figure 1). 
Immobilized S. cerevisiae AXAZ-1 cells were effective at fermenting wort even at very low temperatures ranging from 0 to 5˚C (32 to 41˚F). Fermentation kinetics and routine industrial analyses of resulting beers are shown in Tables 1 and 2. Immobilized cells retained their stability from batch to batch even at very low temperatures and finished fermentation in times ranging from 1 day at 15˚C (59˚F) to 20 at 0˚C (32˚F). At all temperatures the fermentations reached terminal gravity and resulting alcohol levels ranged from 4.4 to 4.9% by volume, showing the effectiveness of this method in low temperature alcohol production. Beer productivities (29–615 g/l/d), and ethanol productivities (1.8–38.7 g/l/d) were high, and proportional to the temperature. The final free cell concentrations were low (0.4–1.6 g/l) and the beers had a fine clarity after the end of primary fermentation. 
Diacetyl and 2,3-pentadione are fermentation byproducts responsible for butterscotch/toffee off-flavors in beer at concentrations above 0.5 mg/L, and lagers require a thorough diacetyl rest to reduce the concentration of these compounds after primary fermentation, which requires high storage capacity, cooling and high energy expenditure. Concentrations of these compounds were measured for each of the DBSG-immobilized beers. At all studied temperatures, the diacetyl and 2,3-pentadione concentrations in the green beers were to low (61–167 ppb and 32–109 ppb, respectively), decreasing with fermentation temperature (Table 2 and Fig. 2). At 0– 5 °C, the 2,3-pentadione content was about threefold lower than that at 15 °C, and at levels similar to those found in mature commercial products (about 30 ppb). Diacetyl in beers produced at 0–5 °C was about half that at 15 °C, which is about double the levels of commercial products (about 30 ppb), but within acceptable levels (<1 ppm).
Dimethyl sulfide (DMS) comes form a malt-derived compound that gets into the wort and gets processed by the yeast during fermentation. It has a very low taste threshold of around 33 ppb and is the primary contributor to the “lager” taste in beer. However, after it reaches a certain concentration, DMS contributes the “cooked veritable” flavors which aren’t so sought after by brewers. In the beers produced with DBSG-immobilized yeast, DMS concentrations varied from 11 to 37 ppm, which is lower than that of most matured commercial lagers (< 40 ppb) and matured beers in general (14-205 ppb). Not surprisingly, DMS concentration was reduces with lower fermentation temperatures and at 0˚C it was three times lower than at 15˚C (Table 2 and Figure 2). 
Fermentation temperatures did not affect bitterness levels (Table 2). Clarity of the beers after primary fermentation was fine and color values matched those of most commercial products.
– Volatiles Analysis: Volatiles were analyzed by gas chromatography (something that’s way, way, WAY out of homebrewers’ or even of commercial breweries’ league). It was found that the total higher alcohol content in the experimental beers was lower than that found in mature commercial products and was reduced as the fermentation temperature decreased. Specifically, 1-propanol, isobutanol and amyl alcohols at 0 °C were reduced by about 41%, 33% and 30%, respectively, compared to those in beers produced at 15°C (Fig. 3). Ethyl acetate concentration was reduced by 30% in 0˚C beers compared to 15˚C. Interestingly, the ratio of higher alcohols and esters remained practically the same at all fermentation temperatures. This means that while higher alcohols’ concentration decreased, so did the fruity ester content. Interestingly, isoamyl acetate (banana-like character) and ethyl caproate (wine-like and fruity character) production was the same between 5 and 15˚C fermentations, but decreased by ~50% when fermentation was carried out below 5˚C. Both were within desired concentration (0.12-0.67 mg/L and 0.07-0.42 mg/L, respectively) and considerably below the “too much” threshold. Acetaldehyde concentrations were stable in green beers at all fermentation temperatures (12.1-18.2 mg/l), which are higher than those found in mature commercial beers (~5 mg/L). 
The group went on to analyze 80 more volatile compounds that are produced during fermentation and influence character of the resulting beer due to their low threshold (Table 4). Most of the identified compounds were esters (mainly ethyl esters of fatty acids and acetic esters of higher alcohols), which are usually found in most beers. The amount of total volatiles was higher in beers produced at 15 °C, indicating higher metabolic activity of the yeast and/or increased chemical transformations at warmer temperature. Miscellaneous compounds, that have relatively low threshold values and usually affect beer flavor with either their fruity, floral or caramel flavors, such as b-myrcene, limonene, linalool, b-damascenone, a-terpineol, anethole, geraniol, phenylethyl alcohol, 3-furaldehyde, 2-furanmethanol, dihydro-5-pentyl-2(3H)-furanone and 4-hydroxy-2-methyl-acetophenone, were also identified. Their amounts were about the same or slightly higher in beers produced at 15 °C, except phenylethyl alcohol and 4-hydroxy-2-methylacetophenone, whose amounts were considerably higher at 15 °C. Off-flavor compounds, such as hexanal (green-leaves odor) and 3-(methylthio)-1-propanol (rotten eggs) were detected in traces in both samples. The sulphur compounds 3-(methylthio)-1-propanol and DMS were found in higher amounts in beers produced at 15 °C. Some compounds such as acetic acid, 8-Nonenoic acid and nonanal were at higher concentration in beers fermented at 0˚C. 
Conclusion:
These results demonstrate that DBSG is an interesting potential cell immobilization substrate for use in brewing and alcohol production industries. It meets the prerequisites for a cost effective industrial application, it is readily available, fully compatible with beer and is of food grade purity from the start. High fiber content gives the grain enough resistivity to stay intact during fermentation. Using cold-loving strain of yeast allowed significant reduction of fermentation time even at very low temperature. Interestingly, young beers fermented at 0-5˚C were characterized by tasters as better, with fine flavor and mature character. This is possibly due to lower concentrations of higher alcohols, diacetyl and DMS as well as reduced content of other volatiles. Tables 3 and 4 show that there is a strong impact of temperature on aroma compound production, though immobilization itself doesn’t affect these processes compared to free cell fermentation as been shown previously in other publication. Fine clarity of the beer right after primary fermentation combined with low vicinal diketone concentrations may possibly lead to elimination of maturation stage, resulting in significant reduction of production time and cost.
My Impressions:
Overall I thought this was an interesting article describing a technology that we may soon see widely available even on homebrewing level. What it comes down to is basically keeping the yeast from freely floating around as the beer ferments and instead keeping it as an easily separable mass within the fermentation vessel while relying on convection to ferment everything out.
Some things that caught my attention:
– The scientists grew yeast in glucose rich medium when getting them to stick to the processed grain before using that grain/yeast complex to ferment wort. This seems to be in direct conflict with the “indisputable homebrewer dogma” that yeast grown with simple sugars can’t ferment your beer well because they get used to processing only simple sugars. I thought about it now and again throughout my homebrewing life and could never really understand it. It isn’t like the genes that encode proteins necessary for breakdown of complex sugars go somewhere. This, of course, doesn’t mean that we should all make table sugar starters, but rather that it’s not such a dramatic effect that you beer won’t ferment out as some homebrew masters will swear to you while, I bet, they’ve never tried to do it themselves. I can see how the health of such culture would get affected due to fast waste buildup, but can’t see how fermentation capacity would be.
– They used a 1:2 – BSG:Wort ratio for their fermentations. That, my friends, is a LOT of grain. For example, if someone is crazy enough to try and do it at home with a 5 gal batch, they’d need about 21 POUNDS of grains with yeast stuck to it. It would be wet and fully saturated so there wouldn’t be any absorption and it would be easily separable from the liquid, but that’s a lot of volume!
– They fermented at temperatures as low as 32˚F! Yes indeed! And those beers apparently turned out really well with low “young lager” associated off flavors. This is very interesting in that this could really eliminate the lagering and diacetyl rest steps and thus save a lot of time and money.
– Low temperature fermentations produce less esters and higher alcohols. That’s not surprising to anyone, but what’s interesting is that psychrotolerant yeast strains can ferment just fine at temperatures so low that even the lager strains stop, fall asleep and precipitate out of solution. In those conditions, ester and higher alcohol production decreases even more, which means the resulting beers would be very malt and hop forward due to very low flavor contributions of the yeast. Just imagine a beer that’s even cleaner than a pilsner! I don’t even know what that would be like, but I’d sure love to find out.
– As with everything in life, there are two sides to this. One example of flaws associated with this method is high levels of acetaldehyde and some other off-flavor compounds that results from such super cold fermentations. That’s a serious concern, but I’m sure it’s only a matter of time before some other psychrotolerant Saccharomyces strain is discovered or even engineered that wouldn’t have these problems and we’ll have very clean and crisp almost-freeze-fermented lagers produced in just a few days.
– This article isn’t the freshest in this line of research, but I chose it because it offered nice explanation of the process and examination of flavor compounds produced during fermentation. I’m sure progress has been made since then and I’ve seen a paper or two on the topic that were as late as 2012 I think. Problem with those papers is that they’re in journals I have no access to. Let me know if you’d like to know more about the topic and I’ll try to dig around and see if there is something interesting to be found.
– Possible future outcome from this line of studies, at least in my view, could be creation of essentially “one-day lagers” that would be fermented in times much shorter than those of normal lagers and would require no or minimal conditioning and diacetyl rest. This would reduce production costs and time, meaning that the breweries would save money. One would hope, perhaps naively, that the saved money would go into something like using malt rather than rice sugar or some other “no flavor, more alcohol” stuff in creating such beers. Perhaps one day we’ll see a full bodied and flavorful, malty and delicious PBR or Coors…
BKYeast 