If data could be gathered more often at a lower cost, bioprocesses would be able to operate at near optimum efficiency.
The biotechnology industry includes products produced by microorganisms such as fuels and chemicals as well as pharmaceuticals and food (for example penicillin, ethanol, acetate, succinic acid, etc.) and plays a significant role in the global economy. Industrial biotechnology relies on living microorganisms that convert a raw material to a product. Throughout this process the microorganisms and their chemical by-products need to be sampled and analysed for quality control purposes.
However, both sampling and analysis are time consuming, labour intensive and costly. This dilemma forces a minimalistic approach to data gathering in order to keep costs down, which in turn can also potentially lead to expensive data misinterpretation due to gaps in information. This challenge provides an opportunity to engage in highly practical and iterative technology development for remote, on-line monitoring to provide real-time data at a low cost.
Shifting a century old paradigm
If data could be gathered more often at a lower cost, bioprocesses would be able to operate at near optimum efficiency. This can be achieved by automated monitoring coupled with automated controls, therefore increasing efficiency further, whilst also driving down operational costs.
In order to accomplish this, a century old paradigm needs to shift. Typically, microbes in bioprocesses have been viewed as chemical catalysts and monitored using chemical and microbiological methods. If there is a disturbance in a bioprocess, this can cause a decrease in microbial activity and therefore effect chemical catalysis. This perturbation will show up eventually in the chemistry and/or microbiology data, but the lag in time due to sample collection, processing and analyses could result in a delayed response to a particular problem.
From a different perspective, the chemical changes in a bioprocess are driven by electron flow, where microbes are the self-replicating catalysts that move electrons that drive reactions converting a feedstock into a product. So, if we think of microbes as electrical circuits as well as chemical catalysts (see Fig. 1) we can employ a whole new arsenal of techniques to follow their activity. These techniques include a wide range of electrochemistry methods such as electrochemical impedance spectroscopy (EIS).
In a simple system, the voltage and current can be used to calculate the resistance (R=E/I). In a more complex system, an AC signal of known frequency and amplitude (Z≡E/I) is included in this calculation where Z is impedance and is a very complex version of resistance.
The change in properties of an AC signal furnishes impedance data that provides a data-rich analytical platform with specific information regarding numerous multifaceted parameters. We use this approach to determine factors such as changes in fluid composition (i.e. conductivity, reaction and diffusion rates), microbial density, metabolic activity, and growth status.1,2,3 Along with our colleagues at the University of South Carolina and Greenway Energy LLC, we are adapting our techniques and equipment to be suitable for work in other labs so that we can collect electrochemical data simultaneously, in conjunction with our collaborator’s data.
Our electrode design can incorporate ‘cyclic voltammetry’ – a technique for determining the electrochemical properties of microbes and their components.3,5 This technique also allows us to clean the electrodes electrochemically.1 Since biofouling of in-situ electrodes is a tremendous problem, our approach permits us to monitor whenever we want without the need to replace electrodes due to biofouling. Voltammetric techniques also provide us with additional analytical capabilities for supplementary chemical interrogation of a system.
Different bioprocesses focus on different growth phases, and it is imperative to know the microbial growth status at any given time for bioprocess optimisation. The ability to acquire real-time data of the growth status of microbes is a significant advantage for a bioprocess to be successful. Additional information can be derived from the electrochemistry data concerning cellular status such as if oxygen concentrations are sufficient or if byproducts may be beginning to inhibit growth and activity.
One of the advantages with impedance techniques is that just one short duration scan (several minutes) of a bioprocess can provide information relevant to numerous parameters. It’s also important to understand that different microbes can produce different impedance responses, based on things like cell size, shape and surface properties. Therefore, each bioprocess should be evaluated on its own physical/chemical characteristics.
Another aspect of biotechnology is environmental cleanup or bioremediation. In this case, microbes (either indigenous or supplemented) are used to catalyse reactions that ultimately degrade carbon-based contaminants (such as petroleum and industrial solvents). Provided they have all the nutrients needed, microbes can destroy these contaminants thereby removing them from the environment. Other contaminants like heavy metals and radioactive elements (i.e. chromium and uranium) can be transformed biochemically by microbes so that these contaminants aren’t a danger to groundwater supplies.
Active bioremediation sites need to be monitored to make sure cleanup is proceeding as predicted. This can be difficult because there are a number of unknowns in any contaminated site that can impede progress. Unfortunately, the costs for sampling in this case are usually much higher than conventional bioprocesses because most often contamination and cleanup activities are deep underground.
Subsurface monitoring can be improved with the electrochemical techniques we are incorporating. Probes placed in-situ for extended periods of time will be able to send data automatically at predetermined intervals. These data can be used to monitor ongoing activity at a cleanup site and provide valuable insight for knowing where and when conventional sampling should take place. In this way a tremendous amount of guess work is removed from the equation and conventional sampling can proceed at specific times and specific locations with a greater certainty of success.
EIS techniques cover a wide range of frequencies for analysis. This permits numerous analytes to be monitored at any given time because specific analytes usually appear in just a portion of the frequencies scanned. While microbial cells may show up in the mid-range of some scans, the biogeochemical changes that the microbes catalyse (i.e. iron mineral transformations) could appear at the very low frequencies. The high density of data produced by this approach will be useful for capturing a big picture of multi-dimensional microbial activity in context with chemical and physical changes that will also be occurring.
The data sent from in-situ electrodes will be integrated mathematically to derive specific parameters from the raw impedance data. Unlike a chemical or microbial sample, the impedance data will not deteriorate over time. Consequently, the raw data can always be integrated for parameters that were not part of the original plan. For instance, if soluble iron begins to show up in chemistry data at a location, previous EIS data can be evaluated to see if low frequency phase shifts show up, implicating iron solubilisation by microbial activity.
In-situ electrodes may also provide a means to monitor the subsurface for changes in the chemistry and microbiology that could be associated with recent sources of chemical contamination. Think of a smoke detector, only underground. With periodic sampling, EIS data can be sent automatically to anyone’s computer providing an update of subsurface activity. This will be extremely useful in areas impacted with potential groundwater contamination from drilling activities or potential leaks from storage tanks. The automated electrochemical cleaning step will minimise analytical problems normally associated with in-situ electrodes.
On the more destructive side, microbial activity, especially biofilms (common surface slime), can cause problems above and below ground associated with metal corrosion and deterioration of wood surfaces and even concrete.6 Biofilms are also very problematic in the medical devise field resulting in new materials and anti-microbial approaches being developed. We are having success at monitoring surfaces using EIS without having to contact the surface.2 With this novel approach the biofilm does not have to be disturbed for it to be analysed. As long as the electrode is positioned at a consistent distance over a submerged surface the detection capability is greater than 10 micro grams of protein per cm2. Rapid and reliable methods are needed in the materials science fields for designing biofilm resistant surfaces. Our EIS approach will provide non-contact analytical capabilities with potential for high throughput sampling.
The advancement of electrochemical techniques and the associated electrical engineering and software innovations offer tremendous potential for exploiting this field for a vast array of inexpensive and rapid bioprocess monitoring techniques as we begin to redefine biotechnology from an electrochemical perspective. Incorporation of microbiology with electrochemical analysis is beginning to open new opportunities for industrial bioprocessing, bioremediation with the expectation of automated bioprocess optimisation in both areas, and rapid high precision evaluation for anti-biofilm material design.
1 Martin, A. L., Satjaritanun, P., Shimpalee, S., Devivo, B. A., Weidner, J., Greenway, S., Henson, J. M., Turick, C. E. (2018). In-situ electrochemical analysis of microbial activity. AMB Express, 8 (162).
2 U.S. Patent No. 10,234,376. Non-contact monitoring of biofilms and corrosion on submerged surfaces with electrochemical impedance spectrometry.
3 Turick, C.E., S. Shimpalee, P. Satjaritanun, J. Weidner, S.Greenway. 2019. Convenient non-invasive electrochemical techniques to monitor microbial processes: current state and perspectives. Appl. Microbiol. Biotechnol. In press.
4 Turick, C. E., Beliaev, A. S., Zakrajsek, B. A., Readon, C. L, Lowy, D. A., Poppy, T. E., Maloney, A., Ekechukwu, A. A. (2009). The role of 4-hydroxyphenylpyruvate dioxygenase In enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1. FEMS Microbiol Ecol, 68, 223-235.
5 Turick, C. E., Ekechukwu, A. A., Milliken, C. E., Casadevall, A., Dadachova, E. (2011). Gamma radiation interacts with melanin to alter its oxidation-reduction potential and results in electric current production. Bioelectrochemistry, 82, 69-73.
6 Turick, C.E., C.J. Berry. (2016). Review of Concrete Biodeterioration in Relation to Buried Nuclear Waste. J. Env. Rad. 151:12-21.
Charles E. Turick
Savannah River National Laboratory