So it has been a pretty intensive 6 weeks of lab work. Ideally I would have liked to keep this blog updated as I was doing the work, however job commitments meant that much of my little spare time has been spent recovering from 18 hour work days. As I am working through my results and writing up my final report I will keep this blog updated regularly with smaller posts relating to what happened chronologically. There are some simple things that could and probably should have been done differently, but this was the first time I have ever done lab work and if others can benefit from my mistakes then I am happy. That being said, there is still plenty to talk about!
As a quick refresher, Chorlton Brewing Company (Manchester) brewed a turbid wort which was then cast to two separate coolship vessels for overnight spontaneous inoculation. The aim of this project was to isolate and identify the yeast and bacteria involved in the fermentation of these beers and assess the viability of the air around a railway arch brewery in Manchester for successful production of lambic-style beer. Sample were taken periodically over three months and stored at 4C until they were sent to me for analysis in May.
The first task was to have a look at the beer under the microscope. It was clearly evident that there were fermentative yeasts and acid-producing bacteria in the beer due to the fact both beers had krausened within two weeks, and that the pH had shown a drop below that of a clean fermentation.
The first stage in identifying the bacteria was using the Gram staining method. This is done by heat fixing a beer sample to a glass slide and staining with crystal violet and counterstaining with safranin-O. Gram positive bacteria (Lactobacillus/Pediococcus) appear as violet under the microscope from being able to retain the original crystal violet stain whereas Gram negative bacteria (Acetobacter/Pectinatus) appear a pink colour due to the safranin-O counterstain and the thin layer of peptidoglycan in their cell wall which prevents retention of the original crystal violet stain. I have some images from a brightfield microscope coming however the (heavily edited) photo above from my phone shows some Gram-positive cocci and maybe the beginning of a chain. I will update with better images when I get them.
I examined the beer samples using a haemocytometer to determine estimated cell counts throughout the fermentation. The aim was to use these cell counts to calculate serial dilutions to obtain 30-300 cells which would then be spread on WLN spiked with chloramphenicol (bacteria inhibitor) to give the viable yeasts cells based on the number of colonies that grew on each plate. Bacterial cell counts were assumed to be 10x that of yeast and so diluted by another factor of 10 and were spread on to WLD (WLN spiked with the antibiotic cycloheximide) to give the total viable bacteria cells.
I’ll have a post in another couple of days with some more specific numbers for the serial dilutions and viable cell counts.
The aim of the wort production for these spontaneous fermentation trials was to produce a wort akin to that produced at Cantillon. By using a modified turbid mashing schedule, enzyme saccharification is controlled to produce a range of short and long chain sugars/starches to provide plenty of carbohydrates for the captured yeasts and bacteria to consume over the duration of the fermentation.
The specific method of wort production was derived from ‘Wild Brews: Beer Beyond the Influence of Brewer’s Yeast’ (Jeff Sparrow, 2005) whilst the grist and hopping rates were derived from Cantillon’s procedure.
- 5.1 Kg Weyermann Pilsner Malt (65% of grist).
- 2.74 Kg Unmalted Wheat (35% of grist).
- 7.84 Kg Total grist weight.
- 140g aged hops (equates to 350 g/hL – based on Cantillon’s method – 25 Kg of hops/brew with a yield of ~70 hL wort into barrels).
- Total vol. of liquor required for rests = 1.9 L Liquor/450 g grain = 34 L
- Initial dough-in liquor:grist = 0.87
- Expected OG = 1.050. Actual OG = 1.052
- Expected FG = 1.004
- Expeted ABV = 6%
- Pilsner and Wheat were mashed in at 64.5˚C with 6.8L hot liquor to reach a mash temperature of 50˚C. Lid was left off mash tun for the first 5 minutes of a 15 minute rest to allow the temperature drop to 45˚C.
- 6.8L of 100˚C liquor added to mash to raise temperature to 52˚C. Rest for 15 mins.
- 4.5L of wort transferred to the kettle and held at 88˚C. The kettle was already primed with 8L of liquor at 88˚C to cover the kettle elements – 12.5L total kettle volume.
- 10L of 100˚C liquor added to mash to raise temperature to 64.8˚C. Rest for 45 minutes.
- A further 10L of wort transferred to kettle and held at 88˚C – 22.5L total kettle volume
- 10L of 100˚C liquor added to mash to raise to 72˚C. Actual temperature hit 74.2˚C so mash was stirred for just under a minute to allow a drop to 72˚C. Rest 30 mins.
- 13L of wort transferred to kettle before all kettle wort was transferred back to the mash to hit a temperature of 76.8˚C. Rest 20 mins.
- Vorlauf to remove large debris.
- Wort cast to kettle and sparged with 88˚C liquor.
- First runnings after vorlauf were at a 1.042SG
- Mash was slightly oversparged – final runnings were at 1.0025SG
- 140g of aged hops were added to kettle and boiled for 2.5 hours to reach a final gravity of 1.052
- Cast to coolships (20 L/coolship).
- One located in brewery.
- One located in carpark.
- Cool overnight.
- Overnight cooling resulted in a temperature of 18.4˚C for the indoor coolship and 15.1˚C for the outdoor coolship.
- Collect cooled wort in fermenters.
Both inoculations had krausened within two weeks with expected drops in pH and gravity – I will have more information on these early stages once i have received further samples as I would like to provide this information as a collective rather than in dribs and drabs. samples from both inoculations have been suspended and observed under a microscope and it appears than the outside coolship captured more yeast than the brewery coolship, however the brewery coolship appears to have captured more LAB. Again i will give more information on this when I have the complete picture.
Once again, a massive thanks to Will and James for powering through the brewday and Will for the fantastic photographs!
So it has recently been confirmed that I am lucky enough to be working in collaboration with one of my favourite breweries, Chorlton Brewing Company, from Manchester! Chorlton was started by Mike Marcus in 2014 and have a focus on producing “contemporary beer inspired by Germanic brewing traditions”. I first came across Chorlton in Cambridge whilst working in a craft beer and gin bar part time to fund my Philosophy bachelors degree. I was new into the world of beer at the time and Chorlton provided me with my first taste of sour beer, opening my eyes to what beer can be.
I remember going home that night and having a look at the infamous Chorlton twitter page run by Mike (https://twitter.com/ChorltonBrew). The honesty and openness resonated with me and, by the time my degree in Philosophy was over I had decided that beer was what I wanted to do.
So it was I ventured to Edinburgh to begin my journey by undertaking an MSc in Brewing & Distilling at Heriot Watt. I had seen that a fairly large percentage of brewers had studied there, saw its fantastic reputation and decided that this would give me the best entry to the beer industry. Had I been unsuccessful in my application I had pieced together a homebrewing kit and would have seen what I could do by going down that route.
Fast forward to September 2017 and I had the chance to meet Mike at the Hanging Bat who were doing a Chorlton tap takeover and meet the brewer event. I was very keen to go in the hope that I would be able to meet Mike and say thankyou for showing me that a business can operate openly and ethically, and for partly inspiring me to choose brewing as a career path.
We met and, with my new course-mates, had a good evening of beers and chat. Come December it was time for me to start thinking about my research proposal for the Summer of 2018. The day before I was due to head back to Edinburgh I emailed Mike proposing a collaborative project looking at spontaneous fermentation having heard in a Beernomicon Podcast that they had trialled brews using this method before. He got back to me straight away and I ended up diverting to Manchester to meet the brewers.
Following some more back and forth emailing between myself and Will, we settled on the idea of attempting two spontaneous inoculations, one inside the brewery and one in the carpark of their railway arch in Piccadilly. The beers will then be transferred to separate FVs in order to compare the proliferation of captured yeasts and bacteria using PCR analysis. I will update this page with further information on specific methods closer to Summer. All images are courtesy of Will from Chorlton.