Biomass Monitoring in Microbial Fermentation

Biomass concentration is defined as the concentration of microbial cells in a culture at a given point in time. Biomass monitoring follows the development of the cell concentration over time and characterizes the growth of the cultivated microorganism.

To describe the development of biomass over time, various parameters (such as backscatter, absorbance, turbidity, optical density (OD), cell dry weight, or cell count) but also synonyms for biomass (like cell density and total cell density or TCD) are used. No matter what it’s called, biomass is one of the most important critical process parameters (CPPs) in microbial upstream bioprocessing.

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Biomass in Bioprocessing

Bioprocessing uses living organisms to convert one or several substrate/s into a desired product. It’s used nearly everywhere today, from the environmentally-friendly production of chemical building blocks to the creation of cutting-edge pharmaceuticals like monoclonal antibodies or cell therapies.

Especially in microbial upstream bioprocessing, where microorganisms such as bacteria, fungi (such as yeast, filamentous organisms), archaea, plants, or algae are cultivated to produce a broad variety of products, biomass is the most commonly monitored bioprocess parameter. In this context it’s mainly applied to suspension cultures, where these microorganisms are grown in microtiter plates, shake flasks, serum bottles, bags, bioreactors, and other cultivation vessels.

Applicable Industries

Food &
Beverage

Fermented Beverages
Animal-free food
Artificial meat
Animal feed

Chemicals
(White Biotech)

Flavors and fragrances
Cosmetics
Fuels
Chemical building blocks
Enzymes

Biopharma
(Red Biotech)

Biopharmaceuticals
Diagnostics

Agriculture
(Green Biotech)

Optimized seeds
Biopesticides

Most microbial growth curves can be broken down into six distinctive growth phases:

Lag Phase: The microorganisms adapt to the surrounding conditions like available nutrients, temperature or pH by adjusting their metabolism. In this phase biomass remains constant as there is no growth.
Acceleration Phase: The organisms start to grow and divide, and the biomass slowly increases. In this phase, the growth rate increases.
Log Phase: The organisms are now fully adapted to the surrounding conditions and grow as fast as possible. The biomass increases exponentially. Growth reaches its maximum rate and then remains constant.
Deceleration Phase: When the primary nutrition source is depleted, the microorganisms cannot grow any further. The growth rate drops rapidly and the biomass increase stops. This phase can be followed by adoption to and growth on another nutrient, if available. If not, the culture will enter the next phase.
Stationary Phase: Without further nutrients, the organisms stop growing. The biomass does not increase any further — the growth rate stays at zero.
Death Phase: After a period of starvation, the microorganisms start to die. The biomass will decrease slowly, which results in a negative growth rate.

Monitor, Control, and Understand Your Bioprocess

Assuming sufficient data density (number of biomass data points over time) and quality, charting the growth curves and kinetics of microbial cultures is crucial for scientists with a range of tasks and goals, including:

Screening for the best strain and or condition(s): When using microbial strains to create a desired product, scientists are concerned with first finding the right strain to generate it, and then with optimizing the conditions so that strain creates the highest possible concentration of the product. They will want to compare the growth curves of various strains and then of the same strain under various bioprocess conditions (such as temperature, media composition, pH, and batch vs. fed-batch).
Timing experimental workflows: Growth experiments often involve various steps (such as inoculation, induction, feeding, sampling, cell or product harvesting, and cooling) where the scientist needs to manually interact with the process. Knowing or, ideally, seeing which growth phase your organism is in helps with the timing of these manual work steps, and increases experimental efficiency and scientific outcome.
Detecting events: Among the important events that may occur during microbial fermentation are diauxic shifts, oxygen-, substrate, product or metabolite-inhibitions, and morphological changes. It’s crucial for scientists to be able to detect these events in order to characterize and optimize both the microorganism and the bioprocess.
Controlling the quality: Following the growth curve can help determine if an experiment is reproducible or not, and if this cultivation can be used for further experiments or production steps. It can also help scientists detect a problem — and avoid costly problems further downstream.

What can happen if you don’t monitor biomass? The biggest pain points scientists report to us are black-box bioprocesses and being “under-sampled”. Without being able to see what happens in the cultivation vessel, and without data, they have no means to screen or time manual workflows, or ensure quality control — and are at a distinct disadvantage. Monitoring biomass ensures projects run smoothly, stay within budget and deliver the expected results.

How Biomass is Measured

Optical Density (OD) Measurements
Backscatter Measurements
Cell Dry Weight Measurements
Capacitance Measurements

Oldie but Goodie

Principle of Measurement: Absorbance

Light with a specific wavelength is emitted into the fermentation broth. A sensor on the other side of the broth detects the intensity of the light as it passes through. The more cells in the culture, the less light. Using the Lambert Beer law, this signal can be used to calculate the optical density, or OD.

Technologies:

Used in photometers combined with offline sampling, in plate readers, or in invasive probes for bioreactors.

Advantages
Disadvantages

MODERN APPROACH, FAST-GROWING TREND

Principle of Measurement: Backscatter

Light with a specific wavelength is emitted into the fermentation broth. A sensor close to the light source detects the amount of the light scattered back by the cells and other particles in the broth. The more cells in the culture, the more light is scattered back.

Technologies:

Used in non-invasive sensors for shake flasks and bioreactors, and in invasive probes for bioreactors.

Advantages
Disadvantages

WIDELY KNOWN BUT SLOW AND TIME-CONSUMING

Principle of Measurement: Cell Dry Weight

Scientists take a defined volume of cells from the cultivation broth and transfer it into a pre-weighed tube. The cells are then dried overnight so that any water is removed and weighed precisely. Subtracting the weight of the empty tube equals the weight of the dried cells per defined volume.

Technologies:

Offline sampling is combined with measuring the cell dry weight on a fine scale.

Advantages
Disadvantages

EXPENSIVE BUT MORE THAN JUST BIOMASS

Principle of Measurement: Capacitance

Microorganisms have a cell membrane, that, if intact, can act as a capacitor when an electric field is applied. The resulting capacitance can be monitored and used to derive information about the cell concentration and the amount of cells with intact membranes — in other words, viable cells.