Abstract: This paper briefly describes the research and application of biosensors, especially microbial sensors, in the fermentation industry and environmental monitoring in recent years, and predicts and looks forward to their development prospects and marketization. Bioelectrodes are sensitive materials that use immobilized biological components as molecular recognition elements. They, along with oxygen electrodes, membrane electrodes, and fuel electrodes, constitute biosensors and are widely used in fermentation industry, environmental monitoring, food monitoring, and clinical medicine. Biosensors have good specificity, are easy to operate, have simple equipment, are fast and accurate in measurement, and have a wide range of applications. With the development of immobilization technology, biosensors have a strong competitive advantage in the market. Keywords: biosensor; fermentation industry; environmental monitoring. Chinese Library Classification Number: TP212.3 Document Identifier Code: A Article Number: 1006-883X (2002) 10-0001-06 I. Introduction It has been 40 years since Clark and Lyons first proposed the idea of ​​biosensors in 1962. Biosensors have received in-depth attention and wide application in fermentation processes, environmental monitoring, food engineering, clinical medicine, military and military medicine. In the first 15 years, biosensors were mainly based on enzyme electrodes. However, due to the high cost and instability of enzymes, the application of sensors using enzymes as sensitive materials was limited. In recent years, the continuous development of microbial immobilization technology has led to the development of microbial electrodes. Microbial electrodes use living microorganisms as molecular recognition elements, which has unique advantages compared to enzyme electrodes. They can overcome the weaknesses of high cost, difficult extraction, and instability. In addition, they can simultaneously utilize coenzymes within microorganisms to handle complex reactions. Currently, the application of fiber optic biosensors is also becoming increasingly widespread. Moreover, with the development of polymerase chain reaction (PCR) technology, DNA biosensors using PCR are becoming more and more common. II. Research Status and Main Application Areas 1. Fermentation Industry Among various biosensors, microbial sensors are most suitable for determination in the fermentation industry. This is because interfering substances often exist during fermentation, and the fermentation broth is often not clear and transparent, making it unsuitable for determination by spectroscopic methods. However, the application of microbial sensors is very likely to eliminate interference and is not limited by the turbidity of the fermentation broth. At the same time, since the fermentation industry is a large-scale production, the low cost and simple equipment of microbial sensors give them a great advantage. (1). Determination of Raw Materials and Metabolites: Microbial sensors can be used to determine raw materials such as molasses and acetic acid, and metabolites such as cephalosporins, glutamic acid, formic acid, methane, alcohols, penicillin, and lactic acid. The basic principle of measurement is to use a suitable microbial electrode and an oxygen electrode. The assimilation of oxygen by microorganisms is utilized, and the amount of oxygen reduction is measured by measuring the change in the current of the oxygen electrode, thereby achieving the purpose of measuring the substrate concentration. Among various raw materials, the determination of glucose is particularly important for process control. The glucose concentration can be estimated by using the metabolism of glucose by Psoudomonas fluorescens and detecting it through an oxygen electrode. Compared with glucose enzyme electrodes, the measurement results of this type of microbial electrode are similar, but the microbial electrode has high sensitivity, good repeatability and practicality, and does not require the use of expensive glucose enzymes. When acetic acid is used as a carbon source for microbial culture, the acetic acid content above a certain concentration will inhibit the growth of microorganisms, so online measurement is required. The concentration of acetic acid can be determined by a microbial sensor composed of immobilized yeast (Trichosporon brassicae), a breathable membrane and an oxygen electrode. In addition, there are also microbial sensors for measuring glutamate, which can be constructed by combining E. coli with carbon dioxide gas-sensitive electrodes, and microbial sensors composed of bacterial-collagen membrane reactors and combined glass electrodes, which can be used to measure cephalosporins in fermentation broth, etc. (2). Determination of total number of microbial cells In terms of fermentation control, a simple and continuous method for directly measuring cell number has always been needed. It has been found that bacteria can be directly oxidized and generate current on the anode surface. This electrochemical system has been applied to the determination of cell number, and the results are the same as those of the traditional plaque counting method [1]. (3). Identification of metabolic test Traditional identification of microbial metabolic types is based on the growth of microorganisms on a certain culture medium. These experimental methods require long culture time and specialized techniques. The assimilation of substrates by microorganisms can be measured by their respiratory activity. The respiratory activity of microorganisms can be directly measured using oxygen electrodes. Therefore, microbial sensors can be used to determine the metabolic characteristics of microorganisms. This system has been used for simple identification of microorganisms, selection of microbial culture media, determination of microbial enzyme activity, estimation of biodegradable substances in wastewater, selection of microorganisms for wastewater treatment, assimilation test of activated sludge, determination of biodegradable products, selection of microbial preservation methods, etc. [2]. 2. Environmental monitoring (1). Determination of biochemical oxygen demand (BOD) The determination of biochemical oxygen demand (BOD) is the most commonly used indicator for monitoring the pollution status of water bodies by organic matter. Conventional BOD determination requires a 5-day culture period, is complicated to operate, has poor repeatability, is time-consuming and labor-intensive, and has a large interference. It is not suitable for on-site monitoring. Therefore, there is an urgent need for a new method that is simple to operate, fast and accurate, highly automated, and widely applicable. Currently, researchers have isolated two new yeast strains, SPT1 and SPT2, and fixed them on glass carbon electrodes to form a microbial sensor for measuring BOD. Its repeatability is within ±10%. When this sensor is used to measure the BOD in pulp mill wastewater, the minimum measurement value can reach 2 mg/l, and the time taken is 5 min [3]. Another new microbial sensor uses yeast strains resistant to high osmotic pressure as the sensitive material, which can work normally under high osmotic pressure. Moreover, its strains can be dried and preserved for a long time, and regain their activity after soaking, providing a quick and convenient method for the determination of BOD in seawater [4]. In addition to microbial sensors, a fiber optic biosensor has been developed to determine the low BOD value in river water. The reaction time of this sensor is 15 min, and the optimal working conditions are 30°C and pH=7. This sensor system is almost unaffected by chloride ions (within the range of 1000 mg/L) and is not affected by heavy metals (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). This sensor has been applied to the determination of BOD in river water and has obtained good results [4]. Now there is a BOD biosensor that has been light-treated (i.e., TiO2 is used as the semiconductor and irradiated with a 6 W lamp for about 4 min), which greatly improves the sensitivity and is very suitable for the measurement of low BOD in river water [5]. At the same time, a compact optical biosensor has been developed to simultaneously measure the BOD value of multiple samples. It uses three pairs of light-emitting diodes and silicon photodiodes. Pseudomonas fluorescens is fixed at the bottom of the reactor with photocrosslinked resin. This measurement method is both rapid and simple, and can be used for six weeks at 4°C. It has been used in the process of industrial wastewater treatment [5]. (2). The commonly used important pollutant indicators for the determination of various pollutants include the concentration of ammonia, nitrite, sulfide, phosphate, carcinogens and mutagens, heavy metal ions, phenolic compounds, surfactants and other substances. At present, a variety of biosensors for measuring various pollutants have been developed and put into practical application. Microbial sensors for measuring ammonia and nitrate are mostly composed of nitrifying bacteria separated from wastewater treatment devices and oxygen electrodes. At present, there is a microbial sensor that can measure nitrate and nitrite (NOx-) under dark and light conditions. Its measurement in a saline environment makes it unaffected by other types of nitrogen oxides. It was used to measure NOx- in estuaries and the results were good [6]. Sulfide determination is achieved using a microbial sensor made from obligate, autotrophic, aerobic thiobacillus thiooxidans isolated and screened from acidic soil near pyrite mines. At pH 2.5 and 31℃, the activity remained unchanged after more than 200 measurements per week, decreasing by 20% after two weeks. The sensor has a lifespan of 7 days, and its equipment is simple, low-cost, and easy to operate. Currently, a photomicrobial electrode is also used to measure sulfide content, using Chromatium SP, connected to a hydrogen electrode [7]. Recently, scientists isolated a fluorescent bacterium in polluted areas. This bacterium contains a fluorescent gene and can produce fluorescent protein under the stimulation of pollution sources, thus emitting fluorescence. This gene can be introduced into suitable bacteria through genetic engineering to create a microbial sensor for environmental monitoring. Luciferase has now been introduced into Escherichia coli (E. coli) to detect arsenic-toxic compounds [8]. The determination of phenol and surfactant concentrations in water bodies has seen significant development. Currently, nine Gram-negative bacteria have been isolated from the soil of the West Siberian oil basin, using phenol as their sole carbon and energy source. These bacteria can enhance the sensitivity of the sensor's sensory component. Its detection limit for phenol is 5 × 10⁻⁹ mol. The optimal working conditions for this sensor are: pH = 7.4, 35°C, and continuous working time of 30 h [9]. Another current-type biosensor for measuring surfactant concentration is made from Pseudomonas rathonis. Microbial cells are immobilized on gels (agar, agarose, and calcium alginate) and polyethylene glycol membranes. They can be cross-linked in the gel using GF/A chromatographic test paper or glutamate-induced cross-linking of microbial cells, maintaining their activity and growth capacity in high-concentration surfactant detection over long distances. This sensor can quickly recover the activity of the sensitive element after measurement [10]. Another current-type biosensor is used to determine organophosphate pesticides, using artificial enzymes. Using organophosphorus pesticide hydrolase, the measurement limit for nitrophenol and diethylphenol is 100 × 10⁻⁹ mol, and it only takes 4 min at 40°C [11]. Another newly developed phosphate biosensor uses pyruvate oxidase G, combined with the CL-FIA desktop computer of the automatic system, which can detect (32-96) × 10⁻⁹ mol of phosphate, can be used for more than two weeks at 25°C, and has high repeatability [12]. Recently, a new type of microbial sensor has been developed, which uses bacterial cells as biological components to determine the content of nonylphenol etoxylate (NP-80E) in surface water. A current-type oxygen electrode is used as the sensor, and microbial cells are fixed on the dialysis membrane on the oxygen electrode. The measurement principle is to measure the respiratory activity of Trichosporum grablata cells. The reaction time of this biosensor is 15-20 min, and the lifespan is 7-10 days (when used for continuous measurement). Within the concentration range of 0.5–6.0 mg/L, the electrical signal is linearly related to the NP-80E concentration, which is well-suited for the detection of molecular surfactants in polluted surface water [13]. In addition, the determination of heavy metal ion concentration in wastewater is also important. A complete monitoring and analysis system for the determination of bioavailability of heavy metal ions based on immobilized microorganisms and bioluminescence measurement technology has been successfully designed. An operon in Vibrio fischeri was introduced into Alcaligenes eutrophus (AE1239) under the control of a copper-induced promoter. The bacteria emitted light under the induction of copper ions, and the degree of light emission was proportional to the ion concentration. By embedding microorganisms and optical fibers together in a polymer matrix, a biosensor with high sensitivity, good selectivity, wide measurement range, and strong storage stability can be obtained. Currently, this microbial sensor can reach a minimum measurement concentration of 1 × 10⁻⁹ mol [14]. There is also a current-type microbial sensor specifically for measuring copper ions. It uses recombinant strains of Saccharomyces cerevisiae as biological elements, which are fusions of the copper ion-inducible promoter on the Saccharomyces cerevisiae CUP1 gene and the Escherichia coli lacZ gene. Its working principle is that the CUP1 promoter is first induced by Cu2+, and then lactose is used as a substrate for measurement. If Cu2+ is present in the solution, these recombinant bacteria can use lactose as a carbon source, which will lead to an change in the oxygen demand of these aerobic cells. The biosensor can measure CuSO4 solution in the concentration range of (0.5 to 2) × 10-3 mol. Various metal ion-inducible promoters have been introduced into Escherichia coli, so that Escherichia coli will show a luminescent reaction in the solution containing various metal ions. The concentration of heavy metal ions can be determined according to the intensity of its luminescence, and the measurement range can be from nanomolar to micromolar, and the time required is 60 to 100 min [15][16]. A biosensor for measuring zinc concentration in wastewater has also been developed. It uses the alkalophilic bacterium Alcaligenes cutrophus and is used to measure the concentration and bioavailability of zinc in wastewater. The results are satisfactory [17]. An algae sensor for estimating pollution in estuarine outflow consists of a cyanobacterium Spirlina subsalsa and a gas-sensitive electrode. The toxicity of water caused by the presence of environmental pollutants is estimated by monitoring the degree of inhibition of photosynthesis. Different concentrations of three major pollutants (heavy metals, herbicides, and carbamate pesticides) were measured using standard natural water as a medium. Their toxic reactions were detected in all samples, and the repeatability and reproducibility were very high [18]. Recently, due to the rapid development of polymerase chain reaction (PCR) technology and its widespread application in environmental monitoring, many scientists have begun to combine it with biosensor technology. There is a DNA piezoelectric biosensor that uses PCR technology to measure a specific bacterial toxin. Biotinylated probes are immobilized on a quartz crystal with a platinum surface containing streptozotoxin antibiotics. Cyclic measurements can be performed on the same crystal surface using 1 × 10⁻⁶ mol of hydrochloric acid. The same hybridization reaction is performed on DNA samples extracted from bacteria and amplified by PCR. The product is a specific gene fragment of Aeromonas hydrophila. This piezoelectric biosensor can identify whether a sample contains this gene, which makes it possible to detect whether various Aeromonas species carrying this pathogen are present in water samples[19]. Another type of channel biosensor can detect toxic substances such as tarantula neurotoxin produced by organisms such as phytoplankton and jellyfish. It is currently possible to measure trace amounts of PSP toxin contained in a single planktonic cell[20]. DNA sensors are also being rapidly applied. Currently, there is a miniaturized DNA biosensor that can convert DNA recognition signals into electrical signals for measuring cryptospores and other waterborne pathogens in water samples. The sensor focuses on improving the recognition of nucleic acids and enhancing the specificity and sensitivity of the sensor, and seeks new methods to convert hybridization signals into useful signals. The current research work is to integrate the recognition device and the conversion device [21]. Microalgin is a bacterial hepatotoxin produced in algal blooms caused by cyanobacteria. A biosensor with a surface cell plasmid genome has been prepared to measure the content of microalgin in water. Its direct measurement range is 50 to 1000 × 10-6 g/l [22]. The idea of ​​using a multiplex biosensor based on enzyme inhibition analysis to measure toxic substances has also been proposed. In this multiplex biosensor, two types of conductors are used—a pH-sensitive electronic transistor and a thermosensitive thin-film electrode, as well as three enzymes—urease, acetylcholinesterase and butyrylcholinesterase. The performance of this biosensor has been tested and the results are good [23]. Besides fermentation industries and environmental monitoring, biosensors are also widely used in food engineering, clinical medicine, military and military medicine, primarily for measuring glucose, acetic acid, lactic acid, lactose, uric acid, urea, antibiotics, glutamic acid and various amino acids, as well as various carcinogens and mutagens. III. Discussion and Outlook Harold H. Weetal in the United States pointed out that the commercialization of biosensors requires the following conditions: sufficient sensitivity and accuracy, ease of operation, low price, ease of mass production, and quality monitoring during the production process. Among these, low price determines the competitiveness of the sensor in the market. Among various biosensors, microbial sensors have the greatest advantages in terms of low cost, ease of operation, and simple equipment, thus their market prospects are enormous and attractive. In comparison, enzyme biosensors are relatively expensive. However, microbial sensors also have their own disadvantages, the main one being insufficient selectivity, caused by the presence of multiple enzymes in microbial cells. There are reports of adding specific inhibitors to solve the selectivity problem of microbial electrodes. In addition, microbial immobilization methods also need further improvement. First, cell viability must be ensured as much as possible; second, the cell-base membrane must be firmly bound to prevent cell loss. Furthermore, the long-term preservation of microbial membranes requires further improvement; otherwise, large-scale commercialization will be difficult. In summary, commonly used microbial electrodes and enzyme electrodes each have their advantages in various applications. If stable, highly active, and low-cost free enzymes are readily available, enzyme electrodes are the ideal choice for users. Conversely, if biocatalysis involves complex pathways, requires coenzymes, or the desired enzyme is difficult to separate or unstable, microbial electrodes are a better option. Other forms of biosensors are also rapidly developing, and their applications are becoming increasingly widespread. With further improvements in immobilization technology and a deeper understanding of organisms, biosensors will undoubtedly open up a new market.