Project Description

What's 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 surfaces^{2, 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 carbon^{5, 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 complex^{5, 10}. The OXC enzyme then catalyzes a reductive reaction in which formyl-CoA and CO2 are produced^{5, 10}. The FRC enzyme then cycles the CoA back to a new oxalate molecule to start the process again^{5, 10}. Thiamine pyrophosphate (TPP), Mg²⁺, acetate, and CoA are required for the reaction to take place^{5, 10}. The reaction mechanism for the breakdown of calcium oxalate by FRC and OXC can be shown by the equations in Figure 1¹⁰:

Oxalobacter formigenes and the Breakdown of Oxalate

In nature, there are several bacteria whose metabolism involves the breakdown of oxalate, a process that could potentially be harnessed to remove beerstone buildup. 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 carbon^5,9. 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 complex^5,10. The OXC enzyme then catalyzes a reductive reaction in which formyl-CoA and CO₂ are produced^5,10. The FRC enzyme then cycles the CoA back to a new oxalate molecule to start the process again^5,10. Thiamine pyrophosphate (TPP), Mg²⁺, acetate, and CoA are required for the reaction to take place^5,10. The reaction mechanism for the breakdown of calcium oxalate by FRC and OXC can be shown by the equations in Figure 1¹⁰: ****INSERT FIG 1 HERE****
Figure 1: Reaction equations for the breakdown of oxalate by Formyl Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC)¹⁰

Calcium oxalate is highly insoluble, due to the strong ionic bond between the calcium and oxalate ions. The calcium oxalate forms a very strong lattice structure as a result of the high coulombic force between the ions¹¹. However, some solubility does occur, reaching an equilibrium with a low ion concentration. As oxalate is broken down into formic acid and CO₂ by the enzymatic process, more calcium oxalate must dissolve into solution in order to maintain equilibrium, as per Le Châtelier’s principle.

Creating a biological system to aid in the Breakdown of Oxalate

Overall given dangers, time and costs to the brewing industry involved in the removal of beerstone, we propose to investigate the use of FRC and OXC for the safe and efficient breakdown of beerstone from brewing equipment. Aside from the production and characterization of FRC and OXC to be used as subsequent treatment on calcium oxalate (and related compounds such as sodium oxalate), we hope to develop a biological system as a form of beerstone treatment. In a cell system, oxalate is brought in by an oxalate-formate antiporter (OxIT) and converted to CO₂ and formyl-CoA. Formyl-CoA is reused by FRC as a CoA donor in a subsequent reaction and released from the cell as formate by OxIT5 (Figure 2).

Utilizing S. cerevisiae, we can transform the episomal plasmid pD1218 into w303α that replaces the MATα pheromone secretion system with our genes of interest (frc, oxc) The OxIT antiporter will also be cloned into our plasmid system to allow for the presence of an alternative energy generation system with both FRC and OXC working to facilitate the breakdown of oxalate. The metabolic activity of w303α will be characterized with a modified Minimum Inhibitory Concentration Assay in which yeast will be grown in varying concentrations of C₂CaO₄ . After 48 hours, the levels of oxalate in solution will be measured using a Calcium Oxalate Dissolution Kit (Trinity Biotech).

****INSERT FIGURE 2 HERE*****
Figure 2. Metabolic pathway of oxalate degradation illustrating the OxIT antiporter⁵

References

1. guelph.ca. Annual & Summary Water Services Report. (2017). at <​http://guelph.ca/wp-content/uploads/AnnualandSummaryWaterServicesReport.pdf​>
2. morebeer.com. Removing & Preventing Beerstone Buildup. (2014). at
3. Gemmel, M. The removal and prevention of beerstone. (2017). at <​http://www.beerlab.co.za/blogs/news/the-removal-and-prevention-of-beer-stone​>
4. Alavi, Z. I. & West, D. B. Proposed Method for the Quantitative Determination of Oxalate in Beer and Wort. ​American Society of Brewing Chemists Journal ​41, 24-27 (1983)
5. Stewart, C. S., Duncan, S. H. & Cave, D. R. Oxalobacter formigenes and its role in oxalate metabolism in the human gut. ​FEMS Microbiology Letters ​230, 1-7 (2004)
6. Johnson, D. & Swanson, H. D. A Look at Biofilms in the Brewery. (2000). at <​http://www.birkocorp.com/brewery/white-papers/biofilms-a-look-at-biofilms-in-the-brewery/​>
7. Paradh, A. D., Mitchell, W. J. & Hill, A. E. ​Occurrence of Pectinatus and Megasphaera in the Major UK Breweries. ​Journal of the Institute of Brewing​ 117, 498-506 (2011)
8. worksafe.vic.gov.au. Cleaning - Using caustic cleaners. (n.d.). at
9. atcc.org. ​Oxalobacter formigenes ​Allison ​et al.​ (ATCC® 35274TM). (2016). At12 <​https://www.atcc.org/products/all/35274.aspx#documentation​>
10. Sidhu, H. ​et al​. ​DNA sequencing and expression of the formyl coenzyme A transferasegene, frc, from​ Oxalobacter formigenes​. ​Journal of Bacteriology ​179, 3378-3381 (1997)
11. Arnott, H., J., Pautard, F. G. E. & Steinfink, H. Structure of Calcium Oxalate Monohydrate. ​Nature​ 208, 1197-1198 (1965)