Team:UofGuelph/Description
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
produced1,2. 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:
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 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:
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).
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 https://www.morebeer.com/articles/removing_preventing_beerstone
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 https://www.worksafe.vic.gov.au/__data/assets/pdf_file/0014/14531/HSS0094_-_Cleaning_-_Using_caustic_cleaners.pdf
9. atcc.org. Oxalobacter formigenes Allison et al. (ATCC® 35274TM). (2016). Athttps://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)
