Industrial microbiology encompasses a broad range of processes and products that harness the activity of microorganisms to remediate waste, produce enzymes and chemical products, ferment foods and beverages, promote health and hygiene, and achieve many other goals. Processes in which the product is the organism itself are common. Genesis Biosciences (Lawrenceville, GA) is one of several companies that offer microbial products—mostly derived from Bacillus species—for cleaning, aquaculture, wastewater treatment, home and business cleaning, and other applications. Products under the company’s Eco-Benign label are “natural” in every sense of the word, consisting of environmentally safe Bacillus spores selected for a particular cleanup task, plant-derived chemicals, and recyclable packaging.

The company’s hard surface cleaner, for example, is formulated from spores that come to life when they encounter environmental triggers such as organic materials. The bacteria then generate extracellular enzymes that break down those contaminants. Similarly, Genesis Biosciences’ domestic animal cleanup products digest not only the obvious unsightly and odiferous components of pet accidents, but also those only the animal recognizes. Bacillus have become the platform species for many such products because they form spores that lend themselves easily to product formulation. Spores resist harsh chemicals and are stable to 65° C. After cultivation in their active state, the microbes are concentrated, spray-dried, and combined with ingredients that promote their stability. “It’s an advantage to use these robust organisms for these applications,” says Amit Choksi, vice president of science and technology at Genesis. “You never know what the conditions will be at a cleanup site, so it’s better to use organisms that can withstand a wide range of conditions.”

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Envera (West Chester, PA) also provides microbes and related services, including strain isolation, microbial selection, process development, large-scale production, and formulation. The company screens organisms—usually Bacillus species—for enzyme production, environmental growth, anti-bacterial and anti-pathogen activity, organic acid production, and plant growth regulation. Its largest production bioreactor is a steam-in-place 6,400-liter fermenter.

“Our bacteria are not used in a transformation process to create a microbially mediated end product. They are instead used for waste digestion, animal feeds, and crop protection,” explains Michael Matheny, president, who describes his company’s main activity as “environmental microbiology.”

Culturing and monitoring

Culture requirements for growing bacteria are simple compared with mammalian cell culture. “Bacillus bacteria scale easily,” Choksi says. Genesis production scales run to 30,000 liters, which is very large by biotech standards. “We’re not as interested in metabolites or secondary products as we are in bioprocessing of therapeutic proteins.”

Downstream purification is also straightforward. Because a Bacillus culture withstands high-heat treatment, after reaching the target biomass and concentration it is simply heated to temperatures that destroy contaminating microorganisms but leave the Bacilli unscathed.

“Hence the boom in Bacillus-based microbiology for bio-ag applications, even in probiotics,” Choksi continues. “Lactic acid bacteria could never withstand the harsh conditions.”

Biomass measurements are critical to industrial microbiology because they indicate optimal harvest time, or the point in a culture’s growth cycle at which microorganisms are optimally fed or induced to enter a final formulation state or to produce products.

Biomass is often quantified by optical density (OD) or absorbance. Classically, microbiologists take optical density measurements spectrophotometrically. A typical method involves dilution to less than 0.5 OD at 600 nm. BugLab (Concord, CA) uses biomass measurement technology that involves light back-scattering through multiple laser and detector pairs, eliminating the need to perform dilutions.

Near-infrared laser light enters the medium, and its interaction with cells scatters the light. Photodetectors collect light that is back-scattered from the cells, the intensity of which is proportional to biomass. Combining results from the multiple laser-detector pairs allows biomass determination over a very wide range, from 0.1 to 300 OD units, without dilution. Back-scattering methods allow non-invasive biomass monitoring, although they cannot distinguish between live and dead cells. Colored media components do not interfere in the near infrared.

The principal strength of optical back-scattering, however, is non-invasiveness. “There is always a chance of contamination after opening a fermenter or shake flask to take a sample,” says Christine Gonzales, BugLab’s sales manager. “There are also issues related to sampling technique, spectrometer performance, and operator error. Non-invasive measurements eliminate those problems.”

Optical reflectance is not the only way to measure cell density or biomass. Aber Instruments (Aberystwyth, UK) and Hamilton (Bonaduz, Switzerland) offer sensors that use radiofrequency impedance to generate measures of a culture’s capacitance or conductivity. Compared to optical techniques, impedance typically has lower sensitivity to biomass, limiting its applicability in the low-biomass range. The benefit to this approach is that it distinguishes live cells, which behave as tiny capacitors, from dead cells, which do not. Combining impedance-based biomass with optical total-biomass measurements can therefore provide information on the health of a fermentation.

Borrowing from traditional biotech

One emerging overlap area between mammalian cell culture and industrial microbiology is the idea of the scale-down experiment. Numerous companies sell benchtop bioreactors in the 1-to-3-L volume range, some of which are suitable for microbial fermentations. These systems have been around for decades. Really catching on, however, are parallel mini-, micro-, and macro-bioreactors with working volumes of between 15 mL and 1 L or more. Established players in this market are TAP Biosystems, Applikon Biotechnology, DASGIP, m2p-labs, Pall, and Pharyx. All provide scale-down and screening systems consisting of parallel bioreactors with differing levels of control for temperature, gas, agitation, etc., and varying degrees of independent operation.

A more recent newcomer, HEL (Lawrenceville, NJ), had previously specialized in chemical reactors and has entered the mini-bioreactor space with its Xplorer HT, which appears to have the widest working volume range capability; independent control of pH and dissolved oxygen, optical density, stirrer speed, and liquid and gas additions; and over-pressure capability. Vessel sizes range from 100 to 500 mL, with working volumes in the 20-to-375-mL range. Larger sizes are optional.

“We’re definitely riding the mini-bioreactor wave,” says HEL’s business development manager, Maurice Knapp. “The established vendors address mostly micro volumes or multi-liter volumes, but companies would like to use mini parallel systems as reliable scale-up models and for troubleshooting scale-down as well. So there’s definitely a market for intermediate volumes.”

Most marketed mini- and micro-bioreactors lack the capability of over-pressure, for example, which is advantageous for enzymatic studies typically run during development of industrial bioprocesses. Enzymes make up a very large slice of industrial micro, where R&D consists of “evolving” microorganisms to produce hardier or more efficient enzyme strains, or investigating conditions under which enzymes work best.

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Because industrial microbiology employs yeast and bacteria instead of mammalian cell expression systems, reactor conditions differ significantly. Fermentations are more rapid, generate more heat, and require more robust agitation and gas delivery, which is why large-scale fermentation-worthy single-use bioreactors have only recently come into their own. Moreover, yeast and bacteria are easier to transfect than cells, and fermentations can lead to quantities of product suitable for testing in hours instead of days. A potential downside to rapid growth and enhanced expression is that engineers have less time to take corrective action if a fermentation goes south.

Preserving the critical characteristics of equipment is essential to microbial scale-up. “The ability to use the same pH and DO sensors used in pilot and production is preferable for any microbial scale-down reactor,” Knapp says. That means port sizes must be identical to those in pilot-scale systems “so customers can use sensors that are common across their entire scale range.”

Microbes require high agitation and oxygenation rates. “They’re often happier under conditions of pressure and temperature in which mammalian cells would die,” Knapp explains. Some microbial studies are involving what he terms “oddball” liquid or gas feeds such as methane, ammonia, corrosive acids, or bases. “That means you have to use stainless steel mini-bioreactors and take special care of filters, tubing materials, and seals,” Knapp adds.

The quality imperative

The focus of the U.S. Food and Drug Administration on process analytic technology (PAT) and quality by design (QbD) has spilled over into industrial biotechnology and microbiology, according to Eric Abellan, product manager for software at INFORS HT (Bottmingen, Switzerland). “We observe the need for faster bioprocess development, especially for our industrial customers who strive for the shortest possible time-to-market,” Abellan tells Lab Manager.

QbD is a risk-based approach that differs from the older practice of testing for quality after the fact, by building quality in during development and, when possible, during production. QbD’s implementation demands understanding of critical process parameters and attributes affecting quality. These are referred to as Quality Attributes (QAs), Critical Quality Attributes (CQAs) and Critical Process Parameters (CPPs).

PAT then becomes the foundation for understanding the relationships between CPPs and CQAs. Briefly, PAT consists of near- and in-process analytics that may be established technologies (such as HPLC) or new approaches to real-time measurements (such as soft sensors, near-infrared, mid-infrared, Raman, etc.), relying strongly on design of experiment (DoE) and multivariate data analysis. DoE is a systematic assessment of multiple factors affecting quality and yield; soft sensors are virtual measurements synthesized through software from other measurements, particularly where hardware sensors are unavailable.

“The key factors are rational process development and a higher understanding of the microbial processes at all scales,” Abellan says.

Next-generation software will centralize much more information coming from various analytical and process monitoring platforms. Additionally, experiment planning will draw from data obtained from completed processes to minimize the number of experiments, thus speeding process development. Current-generation soft sensors provide a rapid way of understanding data generated by hardware sensors during the process,” Abellan explains. Using data from classical sensors—for example, an off-gas analyzer for oxygen and carbon dioxide—a soft sensor can calculate such parameters as respiratory quotient, biomass concentration, and uptake rates for specific substrates. Virtual measurements are subsequently applied for automated process control. “Transfer to larger scale can be much faster,” says Abellan, “thanks to a better understanding of the relevant parameters affected by change of scale.”