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Project Design

Whats the Deal with Beerstone?

Beerstone can form on any surface that comes into contact with beer and wort (unfermented beer) and has been a problem for brewers as long as beer has been produced^{1, 2}. The most problematic locations for its formation are heat exchangers, fermentation vessels, aging tanks, kegs, and beer dispense lines. Beerstone is comprised of a combination of precipitated calcium oxalate and entrapped beer polypeptides³. Oxalate enters the brewing process from the cereal grains and hops used to make beer⁴. Oxalate is present in the form of aqueous oxalic acid which is a corrosive, highly oxidized compound that has strong chelating activity⁵. These oxalate ions are soluble in both wort and beer, allowing them to combine with calcium ions to form calcium oxalate⁴. Calcium ions enter the brewing process through the water, grains and water- correction salts⁴. Calcium oxalate precipitates out of solution upon formation, and is one of the most insoluble metallo-organic compounds with a low solubility⁴. In a geographic region with high calcium levels in the drinking water, such as Guelph, Ontario, this can lead to up to 165g of calcium oxalate building up in a single 1000L batch of beer¹. This precipitation can happen either during the brewing or bottling processes, or after the beer has been bottled depending on when the calcium oxalate formation occurs⁴. The point during the process at which calcium oxalate forms is dependent on temperature, time, pH and ion concentration⁴. If calcium oxalate forms after filtration, or if all calcium oxalate crystals are not filtered out of the beer, haze and sediment may form⁴. In addition, calcium oxalate crystals can cause over-foaming during filling or when a pressurized container such as a can or bottle is opened⁴.

Deposits of beerstone provide protection and nutrients for bacteria to grow due to the porous surface of the beerstone, into which nutrient-providing proteins often become entrapped6. This allows for unwanted microbial growth and the formation of biofilms upon the beerstone, for example, species of the genera Pectinatus and Megasphaera. These microorganisms cause beer spoilage, products with reduced shelf-life, off-flavours and sour tastes, rendering the beer unsuitable for sale or consumption thus resulting in financial loss to the brewer7. Lactobacillus species in beer, in particular, causes high turbidity which manifests as a hazy appearance in the liquid. It also causes a high level of diacetyl in the beer, resulting in an unwanted ‘buttery’ flavour7. With the removal of beerstone, growth of these microbial contaminants will be prohibited, improving brew quality and reducing downstream processing.

Beerstone is difficult to remove for several reasons. Calcium oxalate is extremely insoluble in both hot and cold water, meaning that the use of harsh chemicals is currently required for effective methods of removing beerstone. These involve caustic or other harsh cleaners which are dangerous to work with due to the potential for exposure burns of the eyes and skin, and corrosive damage to surfaces2, 3, 8. The use of these cleaning agents require long and frequent pauses in production which lower the efficacy of the brewing process. Additionally, the equipment required to utilize these caustic chemicals and the chemical disposal requirements are costly and potentially environmentally damaging.

Oxalobacter formigenes and the Breakdown of Oxalate

Oxalobacter formigenes is an anaerobic, Gram- negative bacteria native to the human gut microbiota5. O. formigenes is a safe (biosafety hazard level 1) organism that relies solely on oxalate as its source of energy, as well as its main source of carbon5, 9. Metabolism of oxalate is accomplished by two enzymes, Formyl Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC)5, 10. In the first reaction step, a Coenzyme A (CoA) is transferred to oxalate by the FRC enzyme10. It functions by forming a ternary complex between the substrates and the enzyme, resulting in an oxalyl-CoA complex5, 10. The OXC enzyme then catalyzes a reductive reaction in which formyl-CoA and CO2 are produced5, 10. The FRC enzyme then cycles the CoA back to a new oxalate molecule to start the process again5, 10. Thiamine pyrophosphate (TPP), Mg2+, acetate, and CoA are required for the reaction to take place5, 10. The reaction mechanism for the breakdown of calcium oxalate by FRC and OXC can be shown by the equations in Figure 110:

Our Project Design

Here we will include information about why we plan on doing our project the way we are, what our plans are in some detail, and why we are doing what we are doing this year

To be, or not to be: that is the question: Whether ’tis nobler in the mind to suffer The slings and arrows of outrageous fortune, Or to take arms against a sea of troubles, And by opposing end them? To die: to sleep; No more; and by a sleep to say we end The heart-ache and the thousand natural shocks That flesh is heir to, ’tis a consummation Devoutly to be wish’d. To die, to sleep; To sleep: perchance to dream: ay, there’s the rub; For in that sleep of death what dreams may come When we have shuffled off this mortal coil, Must give us pause: there’s the respect That makes calamity of so long life; For who would bear the whips and scorns of time, The oppressor’s wrong, the proud man’s contumely, The pangs of despised love, the law’s delay, The insolence of office and the spurns That patient merit of the unworthy takes, When he himself might his quietus make With a bare bodkin? who would fardels bear, To grunt and sweat under a weary life, But that the dread of something after death, The undiscover’d country from whose bourn No traveller returns, puzzles the will And makes us rather bear those ills we have Than fly to others that we know not of? Thus conscience does make cowards of us all; And thus the native hue of resolution Is sicklied o’er with the pale cast of thought, And enterprises of great pith and moment With this regard their currents turn awry, And lose the name of action.–Soft you now! The fair Ophelia! Nymph, in thy orisons Be all my sins remember’d.

Future Development

Here we will write about our plans for the future of this project and what direction we plan on heading.

To be, or not to be: that is the question: Whether ’tis nobler in the mind to suffer The slings and arrows of outrageous fortune, Or to take arms against a sea of troubles, And by opposing end them? To die: to sleep; No more; and by a sleep to say we end The heart-ache and the thousand natural shocks That flesh is heir to, ’tis a consummation Devoutly to be wish’d. To die, to sleep; To sleep: perchance to dream: ay, there’s the rub; For in that sleep of death what dreams may come When we have shuffled off this mortal coil, Must give us pause: there’s the respect That makes calamity of so long life; For who would bear the whips and scorns of time,

References

Rose, D. This is a test (2017). Sci. Awesome. 28-29