Everything for Electronics

DIY Biotech: Harnessing Bacteria for Fermentation
Nuts & Volts Magazine (November 2018)

DIY Biotech: Harnessing Bacteria for Fermentation

By Bryan Bergeron    View Digital Edition  


Photo 1. (Above) Lactobacilli under the microscope are rod-shaped bacteria that stain Gram-positive (purple/violet).

In this first installment of a series of DIY Biotech articles, we’ll look at bacterial fermentation in the form of yogurt making. Not only is this a low-cost, low-risk entry into practical DIY biotech, you’ll learn a lot about monitoring pH as well.

INTRODUCTION

As noted in my editorial in the September-October issue, “DIY Biotech: The Ultimate STEM Track,” DIY Biotech is hot. It’s affordable, accessible, and — thanks to the readily available gene editing tool, CRISPR — amazingly powerful. Putting aside the real possibility of scenarios depicted in Rise of the Planet of the Apes and Gattaca, it’s important to note that we’ve been harnessing the power of bacteria to modify and preserve our food since before written history.

Take yogurt, for example. Thanks to a few beneficial bacteria such as Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus acidophilus, milk that would otherwise spoil can be transformed into a creamy tart treat that can be saved for days without refrigeration.

On the surface, making yogurt couldn’t be simpler. I can still remember watching my great-grandmother prepare yogurt. Before going to bed, she’d put an enameled pan on the gas stove, half fill it with whole non-homogenized milk, and quickly bring just the edges of the milk to a boil. She’d then move the pan to the sink, which was filled with about two inches of cold tap water.

After a few minutes, she’d move the lukewarm milk — as measured by her finger — to the countertop. Next, she’d add a tablespoon of yogurt from a previous batch and stir. She would then move the pan to the warm stove top just over the pilot light, cover the pan with a plate, and turn out the lights.

In the morning, she checked the yogurt for taste and consistency. If the yogurt was tart and firm, she’d put it in the refrigerator to use at lunch and dinner. On the rare occasion the yogurt was bland or runny, she tossed the batch and sought out some starter — yogurt with viable bacteria — from a neighbor.

The genesis of my great-grandmother’s starter — and presumably the neighbor’s — was a teaspoon of rain water from the first shower in April.

DIY yogurt making hasn’t changed much over the decades. Sure, there are yogurt makers that provide a specific temperature over a set time period, and most kitchens are equipped with thermometers. Many cooks prefer a freeze-dried starter over a teaspoon of yogurt or rainwater. However, the basic bacterial process that converts milk to yogurt — fermentation — predates cows and milk, and even the dinosaurs.

Fermentation may be ancient, but if you really understand the significance of each step, you’ll be primed to master even the most complex modern genetic manipulation techniques.

Now, let’s make some yogurt using modern DIY biotech monitoring and production methods to insure a safe, quality, and consistent product.

THE SCIENCE

Key STEM concepts include:

  • Fermentation is an anaerobic (oxygen free) process used by bacteria to extract energy from sugar.
  • Fermentation of Lactose by Lactobacillus produces lactic acid as a waste product.
  • Lactic acid preserves, thickens, and flavors the milk, while inhibiting bacterial growth.
  • Fermentation is best monitored by measuring pH.

FERMENTATION

The various species of Lactobacilli live in and all around us: in the soil, in our digestive systems, on plants, in rain water, and, of course, in milk. Fermentation is a digestive process used by bacteria to extract energy from food. In the case of Lactobacilli, the food is lactose or milk sugar.

Lactobacilli produce lactase, an enzyme that breaks down lactose into two simpler sugars: glucose and galactose. These sugars are taken into the Lactobacilli where they undergo glycolysis. This breaking down of glucose and galactose produces energy that the Lactobacilli need to grow. Lactic acid is what’s left of the simple sugars once they’ve been used up. All this occurs in an anaerobic environment — that is, without oxygen.

As lactic acid levels increase during fermentation — from 6 to about 4 — the environment becomes less hospitable to bacterial growth. This is a good thing because many bacteria are harmful. Take Clostridium botulinum — as in botulism — which can’t grow in a pH below 4.6 [1]. Eventually, even the growth of Lactobacilli (an acid tolerant bacteria) is self-limited by the acidic environment.

pH AND ACIDITY

pH — the power of Hydrogen — is a measure of hydrogen ion (H+) concentration relative to hydroxide ion (OH-) concentration in a solution. The more acidic a solution, the greater the relative concentration of hydrogen ions. Monitoring pH is important in water purification, in soil analysis, in food science, electroplating, in the paper industry, and in the pharmaceutical industry, among others.

The pH scale which is logarithmic to base 10 (hence, the power of hydrogen) ranges from 0 to 14 — or acid to base. A pH of 7 — the pH of water — is neutral. Our bodies are somewhat alkaline, with a pH of around 7.3. Because the pH scale is logarithmic, a yogurt culture with a pH of 5 is ten times more acidic than a culture with a pH of 6. As such, even a small shift in pH (say, from 4.6 to 4.4) is a significant increase in acidity. This decrease in pH of only 0.2 is equivalent to a doubling in acidity:

  • Acidity = 10 log (104.6 – 104.4)
  • Acidity = 10 log (100.2)
  • Acidity = 2

An inexpensive means of measuring pH is to use pH paper or strips. A strip of paper is treated with different pH indicators and allowed to dry. When part of the strip is dipped into a solution, the indicators change color as a function of the solution’s pH.

Some pH paper is designed to work with the entire 0-14 range, while others are restricted to a smaller range. When working with yogurt, only a small part of the acidic range is of interest.

Ideally, the pH paper would change color significantly over the range of 4-5. In practice, the nearest commercially available range is used; in our case, 3.0-5.5. One problem with pH paper — especially for users with some form of color blindness (10% of males suffer from color blindness) — is correctly matching the colors on the reference scale to the color of the test strip.

Digital pH meters (while a pain to work with) can be very accurate and easy to read. Digital (and analog) pH meters work by measuring the current that flows between two specially constructed electrodes when the electrodes are immersed in a solution. An ion exchange occurs when the glass electrode containing a solution of potassium chloride at a pH of 7 is immersed in a moderately acidic yogurt culture (Figure 1, left).

Figure 1. pH electrodes immersed in moderate (left) and more (right) acidic yogurt. H+ ions shown in yellow.


Different amounts of ion swapping occur on the two sides of the glass, creating a voltage differential between the internal electrode and the external reference electrode. The greater the acidity of the yogurt culture (Figure 1, right), the greater the ion/voltage differential. This voltage differential is amplified and applied to an A-to-D (analog-to-digital) converter and displayed by the pH meter.

DENATURED PROTEIN

Another effect of increased lactic acid concentration due to fermentation is denaturing of the milk proteins. Milk proteins normally exist as tight bundles of amino acids that minimally interact with other proteins. An acidic environment causes the milk proteins to unfold and become entangled. As a result, the milk gels and takes on a custard-like texture.

Heat also denatures milk protein and the protein within bacteria. Milk sold commercially in the US is pasteurized (heat treated) for several seconds at temperatures well above 212°F/100°C. Even though the majority of live bacteria in a batch of milk may be killed by pasteurization, their spores remain. Bacterial spores (think seeds or escape pods) aren’t affected by processes that would leave the milk protein intact. As a result, pasteurized milk isn’t sterile, and must be heat treated again in the yogurt making process.

EXPERIMENTAL DESIGN

When it comes to making yogurt at home, you have options. There’s the full DIY approach used by my great-grandmother, and the no-brainer approach of using one of the dedicated yogurt makers where you simply add starter and milk to a solution, fill some cups with the mixture, and wait a few hours. Despite the popularity of time-based yogurt makers, commercial producers moved from a time- to a pH-based approach because of inconsistent results.

In this experimental design, we’ll compare the results of traditional time-based yogurt production such as a recipe from the New York Times [2], with a method based on lactic acid production as monitored by the pH of the fermenting milk. The ingredients are identical.

The key variable is how to determine the end of fermentation. Use the minimum time to completion specified in your favorite recipe or automatic yogurt maker. I’ve seen advertisements of yogurt in as little as 4.5 hours for yogurt makers. For the pH-based experimental group, we’ll halt the fermentation process once the pH drops to 4.3. We’ll compare the pH of our experimental yogurt with that of time-based fermentation, with the expectation that the pH and texture/taste will be different. These differences could be due to variations in unwanted bacteria and milk sugar content.

EQUIPMENT REQUIRED

  • Clean/sterile containers and utensils
  • Thermometer
  • pH meter or pH paper

You’ll need a collection of clean — and optimally sterilized — pots, containers, stirring spoons, and a thermometer or a commercial yogurt maker. You’ll need the same basic utensils for the pH-based fermentation group, but no yogurt maker. However, if you have access to clean beakers and mixing flasks, then you should use them. This is especially true if you need your experiment to look “scientific” as part of a STEM project for school.

You’ll need some way to sterilize or at least clean everything. I use a Presto 1755 16 quart aluminum pressure cooker/canner (Amazon, $77), my DIY autoclave, or sterilizer.

You’ll need a good thermometer. Many yogurt makers come with thermometers that can’t be trusted. I like to use my Fluke 62 Mini IR thermometer ($50, eBay) simply because it provides a fast estimate of the current temperature and it’s one less item I need to keep sterile (see Photo 2).

Photo 2. Fluke 62 Mini IR thermometer taking measurement of milk temperature.


If you need to buy a contact thermometer, then consider the Thomas Traceable Long Stem Digital Thermometer ($45, Amazon). It’s accurate and tracks changing temperatures quickly. A glass lab thermometer ($12, Amazon) also works well, although I find them slow to respond to temperature changes and difficult to read at off-angles.

Then, there’s the pH monitor. You have two choices based on your budget. The first is an inexpensive roll of pH monitoring tape (Amazon, $12). As shown in Photo 3, look for the limited range pH paper instead of the usual 0-14.

Photo 3. Limited range pH paper showing the pH of fresh milk, which is above 5.5.


If you can afford it and have a little patience, then you should consider a pH meter. The meter probe should be designed for liquids. Photo 4 shows the pH meter I used for this experiment: the temperature-compensated Extech pH100 ($80, Amazon). The unit is a compromise between affordability and accuracy.

Photo 4. Extech pH100 and calibration solutions.


There are pH meters with probes designed expressly for commercial yogurt production, but they’re expensive ($395, Hanna, Inc.). Regardless of the price, pH meters are finicky creatures, and you’ll need calibration solutions like those shown in Photo 4, if you want accurate, repeatable measurements.

To calibrate the pH meter, you’ll need two buffers or standards with a pH that borders the monitoring range of our experiment. Given the expected working range of the experiment, I picked up the General Hydroponics pH 4.01 and pH 7.0 Calibration Solution Kit, 8 oz. ($15, Amazon). Calibration involves immersing the electrodes in the 7.0 solution, hitting the calibration button until the unit signals OK, and repeating with the pH 4.01 solution.

Using the pH meter is as simple as immersing the clean probe into a sample of the milk solution and pressing the button. Although tempting, avoid plunging the probe tip into the main container of yogurt. The probe tip isn’t sterile.

CONSUMABLES

  • Whole milk
  • Commercial yogurt with live cultures/freeze-dried starter
  • Deionized or distilled water

For the control (time method) and experiment (pH method) batches of yogurt, you’ll need pasteurized whole milk and a starter culture. The amount of milk you need depends on the capacity of your pots/yogurt maker/other containers.

For the starter, you can use a teaspoon of fresh commercial yogurt that you’ve allowed to reach room temperature — as long as it has “live cultures” listed on the label. If you want to avoid ruining a batch because of contaminated or weak yogurt-based starter, you can use a commercial dried starter that’s guaranteed to contain a specific microbe mix. I used Yogourmet Freeze-Dried Yogurt Starter ($7 for six 5g packets, Amazon).

THE EXPERIMENT

To make the most of this learning experience, treat it as a standard laboratory exercise. That means wash up and wear a lab coat, examination gloves, and eye protection. Until you get to the taste test, work as though the yogurt contains pathogenic bacteria.

1. Sterilize the equipment.
At a minimum, boil the utensils and containers. Because many bacteria and virtually all spores will survive mere boiling water at 212°F/100°C, consider using a pressure cooker to steam your containers at a higher temperature and pressure. For this experiment, I steamed all containers and utensils at 15 lb and 250°F/121°C for 15 minutes. See Photo 5.

Photo 5. Pressure cooker setting for 15 minutes at 250°F/121°C.

2. Rapidly heat the milk to at least 180°F/82°C.
This step helps reduce the viability of some of the harmful bacteria in the milk. Photo 6 shows my hot plate stirrer ($70, eBay) and Thomas Traceable Long Stem Digital Thermometer in action.

Photo 6. Checking the temperature of milk on a hot plate stirrer.

3. Rapidly cool the milk to 110°F/43°C.
For this step, place the pot or beaker with the hot milk into a pan or sink partially filled with cold tap water. You can put ice in the water bath after a minute or two to speed the cooling process.

4. Pour off a quarter cup of milk from the main container and mix it with the starter yogurt (Photo 7).
If you use a commercial powdered starter, then follow the mixing directions on the starter package.

Photo 7. Pour off milk for mixing with the starter.

5. Inoculate the large container of warm milk with the starter.
Stir the starter mixture or powder into the warm milk. At this point, you should have two batches of yogurt ready for fermentation.

6. Measure the pH of the experimental mixture.
Use a piece of pH paper or a calibrated pH probe to establish the initial pH of the experimental mixture. Use a clean/sterile cup to take a sample of the mix from the main container for testing. There’s no need to measure the pH of the other batch — the pHs should be identical. The initial pH in my yogurt batch was 6.42, as shown in Photo 8.

Photo 8. Measuring the pH of milk before fermentation.

7. Move the milk to an incubator set to 110°F/43°C.
Lacking an incubator, find a warm place to store both batches of yogurt. If you’re using a yogurt maker, then follow the instructions for incubation.

8. Measure the pH of the experimental mixture at two-hour intervals.
Measure the pH of the experimental mixture at two-hour intervals until the pH falls to 4.3, as in Photo 9. Refrigerate immediately. Remove the other control batch of yogurt from the yogurt maker or incubator at the minimum specified time to completion and refrigerate. If you’re following the New York Times recipe, then the minimum time to refrigeration is six hours. If you’re using the Yogourmet Multi Yogurt Maker, then it’s only 4.5 hours.

Photo 9. Yogurt pH at the end of the process; about 4.3.

9. Compare the pH of the yogurts made through time- and pH-monitored fermentation.
Compare the pH of the yogurts made through the different processes. Note the consistency and — if you dare — the taste of each yogurt. The taste test is a no-no in an academic or industrial lab, but at home, you have the final say.

OUTCOMES

In my experiment, the New York Times recipe resulted in yogurt with a pH of only 4.7 — not acidic enough to kill some pathogenic bacteria and not tangy enough for my taste. It took an additional six hours of fermentation at 110°F/43°C to reach a pH of 4.3. At the same pH, both batches of yogurts were smooth and creamy.

ANALYSIS & DISCUSSION

The pH of the yogurt made using the time-dependent recipe failed to drop below 4.3 in the six hour minimum incubation time. This may have been due to the ambient temperature, pressure and humidity, the amount of lactose in the milk, or some other factor(s). This is precisely the reason for using pH to determine when to end the fermentation process.

You might be wondering why we don’t simply allow the yogurt to ferment for 24 or more hours, just to be safe. One issue is time. Who wants to wait all day?

Another is the quality of the yogurt. If the fermentation is allowed to progress beyond a certain point, then the yogurt becomes excessively acidic and separates into runny clumps.

If you’re reading this article with a STEM project in mind, then consider verifying that the bacteria used in the starter actually ends up in the final product. You’ll need agar plates to visualize the colonies and a microscope and various stains to see the individual bacteria.

If you’re unfamiliar with agar plates and stains, don’t worry. We’ll get there in this series. For now, let’s take baby steps and get acquainted with a few micro-organisms.

Future articles in this series will continue the exploration of fermentation using a different life form and a new set of monitoring technologies. We’ll take a close look at some lab equipment, and make some colorful bacteria using CRISPR.  NV


REFERENCES

1. Clostridium botulinum. USDA Food Safety Inspection Service; https://www.fsis.usda.gov/wps/wcm/connect/a70a5447-9490-4855-af0d-e617ea6b5e46/Clostridium_botulinum.pdf?MOD=AJPERES. Accessed Aug 1, 2018.

2. Creamy Homemade Yogurt. Melissa Clark; Cooking; nytimes.com. Accessed Aug 1, 2018.




Comments