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Review

Application of Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil

by
Kai Zhang
*,
Meng Liu
,
Xinlong Song
and
Dongyu Wang
School of Chemistry and Environment, China University of Mining and Technology (Beijing), Beijing 100083, China
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(9), 7351; https://doi.org/10.3390/su15097351
Submission received: 31 March 2023 / Revised: 24 April 2023 / Accepted: 26 April 2023 / Published: 28 April 2023
(This article belongs to the Special Issue Farmland Soil Pollution Control and Ecological Restoration)
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Abstract

:
The luminescent bacteria bioassay has been commonly used in the detection of environmental pollutants. Compared with traditional chemical and other biological detection methods, the luminescent bacteria bioassay has many demonstrated advantages such as a sensitive response, low cost, high efficiency, and environmental friendliness. The traditional luminescent bacteria bioassay has poor reproducibility and cannot achieve undisturbed soil testing, and the use of leach liquor also affects the results. This paper reviews the research progress and existing issues for the traditional luminescent bacteria bioassay used in the detection of soil pollutants. The luminescence mechanisms and detection principles of three commonly used luminescent bacteria, i.e., Vibrio fischeri, Photobacterium phosphoreum, and Vibrio qinghaiensis, are discussed and compared. In addition, two new luminescent bacteria bioassays are introduced to detect soil pollutants. One method is based on recombinant luminescent bacteria obtained with a gene-modification technique. This method can realize specific detection and enhance sensitivity, but it still cannot achieve undisturbed soil detection. The other method involves using magnetic nanoparticle (MNP)-based biosensors made from luminescent bacteria and MNPs. It can realize the accurate detection of the biological toxicity of the combined pollutants in soil without disturbing the soil’s integrity. This study shows that MNP-based biosensors have good application prospects in soil pollution detection, but the mechanism behind their utility still needs to be investigated to realize their popularization and application.

1. Introduction

In recent decades, soil pollution has become increasingly serious with the development of industry and agriculture [1]. Soil is an important resource on which people rely for survival and development, so related researchers need to establish effective detection methods of soil pollution and provide fundamental support for efficient treatment strategies [2].
The majority of the early technologies for detecting soil pollution were chemical ones. Spectrometry and chromatography are two examples of frequently used chemical techniques [3,4,5]. By employing optical tools such as spectrophotometers to analyze the composition of soil, spectrometry may both qualitatively and quantitatively identify contaminants. Chromatography is a technique for separating and examining contaminants by taking advantage of the variations in distribution coefficients between the stationary and mobile phases of various substances. Liquid chromatographs and other detective instruments are frequently employed. However, chemical detection methods can only measure the content of pollutants, but cannot directly reflect the biological toxicity [6,7,8]. Additional in vivo and in vitro experiments or models are usually needed to assess the toxicity of the pollutants [9,10]. In addition, specific chemicals such as diethyl triamine pentaacetic acid (DTPA)and hydrochloric acid (HCl) are usually added to process the soil samples in traditional chemical methods, which can affect the physical and chemical properties of pollutants [11]. In particular, when there are synergetic, impedance, and other effects due to multiple pollutants, models cannot accurately and quantitatively evaluate the comprehensive biological toxicity [12].
Therefore, the application of bioassays that can directly reflect the toxicity of single or multiple pollutants in soil by detecting the systematic biological response of certain organisms to pollutants is beginning to become more widespread [13]. Fish, algae, daphnia magna, and luminescent bacteria are frequently used in bioassay methods to perform a toxicity test [14]. However, it is time consuming and expensive to use fish, algae, and daphnia magna in a bioassay [15]. Compared with bacterial growth inhibition experiments and enzyme activity experiments in microbial toxicity experiments, the results of bacterial growth inhibition experiments are more accurate, but the operation is more cumbersome [16]. The enzyme activity experiment has good stability, but it takes a long time. The luminescent bacteria method is easier to operate and responds quickly [17]. Thus, it has received increasing attention in toxicity detection [18].
The luminescent bacteria bioassay can intuitively reflect the toxicity of pollutants in luminescent bacteria with high sensitivity [19]. Heisterkamp et al. used algae, water fleas, fish eggs, and luminescent bacteria to analyze the biotoxicity of leachate from reactive flame-retardant coatings [20]. HeDeFu et al. used fish, xenopus tropicalis embryos, and luminescent bacteria to evaluate the ecological toxicity of water from heavily polluted rivers [21]. Gartiser et al. used algae, zebrafish, daphnia magna, and luminescent bacteria to detect the eluate ecotoxicity of building material HSR-2 (roof sealing sheet) [22]. All the results show that luminescent bacteria are more sensitive than algae, daphnia, and zebrafish in detecting pollution caused by heavy metals and organic matter. In addition, the luminescent bacteria bioassay can also be used to detect the biodegradation effect of pollutants [23]. The chemical decomposition of microorganisms is used to transform the pollutants into carbon dioxide and other inorganic compound forms so that they can efficiently degrade pollutants or reduce the toxicity of pollutants [24,25].
However, there are some problems with the traditional luminescent bacteria bioassay in detecting soil contamination. The turbidity of the soil affects the capture of luminous intensity [26]. Additionally, traditional centrifugal extraction and other operations affect the physical and chemical properties of the soil. Therefore, based on the traditional luminescent bacteria bioassay, two new detection methods are proposed. One involves using genetic engineering to transfer the structural genes responsible for luminescence to other bacteria and assemble them into recombinant luminescent bacteria, which can realize the detection of specific pollutants [27,28]. The other is to use magnetic nanomaterials and luminescent bacteria to assemble magnetic nanoparticles (MNPs)-based biosensors, which can be separated from the soil solution and tested under an external magnetic field, without centrifugation and other operations, to realize the detection of undisturbed soil [29].

2. Data Source Search Methods

A literature search was used to access relevant material in this review. We comprehensively searched the Web of Science database and CNKI database from 2014 to 2023 using the following terms: “Luminescent bacteria” or “Vibrio fischeri” or “Photobacterium phosphoreum” or “Vibrio qinghaiensis” or “Recombinant luminescent bacteria” or “Luminescent bacteria method and soil” or “magnetic nanoparticles and bacteria”. No restrictions were set on language, document type, or data category. Preliminary selection was conducted based on the titles and abstracts of the papers. Secondary screening was conducted by checking the details. A total of 110 publications were selected. Three commonly used luminescent bacteria (Vibrio fischeri, Photobacterium phosphoreum, and Vibrio qinghaiensis) were investigated for their detection and application in contaminated soil. Studies to improve the traditional luminous bacteria method were also prioritized. The application of the traditional luminescent bioassay in the detection of contaminated soil was summarized. Two new research hotspots were determined to achieve biological toxicity detection of undisturbed soil. The detection of genetically engineered luminescent bacteria could achieve specific detection, and MNP-based biosensors could achieve the detection of undisturbed, contaminated soil. Future research directions were also proposed to solve the reproducibility problems of MNP-based biosensors in practical applications.

3. The Application of a Traditional Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil

Initially, a luminescent bacteria bioassay was used in the water environment [30,31]. Photobacterium phosphoreum T3 has been used to determine acute toxicity of water pollutants as a national standard since 1995 [32]. With the increasing application of the luminescent bacteria bioassay in the water environment, studies have also been conducted to apply the luminescent bacteria bioassay in contaminated soil systems [33].
Luminescent bacteria can emit blue-green visible light with a wavelength of 450–490 nm, and the luminous intensity is constant under certain stable conditions [34]. The luminous intensity of luminescent bacteria is inhibited when the bacteria contact a certain toxicant. Additionally, the degree of inhibition is correlated with the concentration of the toxicant [35]. In the luminescent bacteria bioassay, the three most-used bacteria are Vibrio fischeri (presently Aliivibrio fischeri), Photobacterium phosphoreum, and Vibrio qinghaiensis [36]. Among them, the European Union standard ISO 11348-3 uses Vibrio fischeri as a standard for assessing wastewater, aqueous extracts and leachates, fresh water, sea water, brackish water, etc. [37]. The Chinese standard GB/T 15441-1995 widely uses phosphorescent bacteria in a luminescent bacteria method for the determination of acute toxicity in the water environment. This standard is applicable to the detection of acute toxicity in industrial wastewater, wastewater receiving water bodies, and soluble chemicals under experimental conditions. There is no standard that uses Vibrio qinghaiensis, but Vibrio qinghaiensis shows a great advantage in testing as freshwater bacteria [38]. The three luminescent bacteria are applied in different conditions [39,40,41], as shown in Table 1.
Although there are differences in the applicable conditions in Table 1, the luminescence mechanism of the luminescent bacteria is similar (Figure 1) [42].
The molecular oxygen in luminescent bacteria reacts with reduced flavin mononucleotide (FMNH2) and long-chain fatty aldehyde (RCHO) with luciferase as a catalyst, and releases blue-green light with a wavelength of 450–490 nm [43]. Thereinto, the luciferin gene (lux) is widely expressed in luminescent bacteria and mainly includes luxC, luxD, luxA, luxB, luxE, and luxG. LuxA and luxB encode the enzyme luciferase, and this enzyme comprises two subunits, αand β, which have high specificity for FMNH2 in the luminescence process. LuxC, LuxD, and LuxE encode acyl protein reductase, acyltransferase, and synthetase, respectively. The multienzyme can help form RCHO. LuxG mediates electron transfer. Nicotinamide adenine dinucleotide (NADH) is used as an electron donor to transfer hydrogen ions in NADH to flavin mononucleotide (FMN) and convert them into NAD+ and FMNH−, providing FMNH− for the reaction [44].
In addition, the three kinds of luminescent bacteria introduced above have similar detection mechanisms for pollutants, and all use the non-specific luminescent bacteria to detect contaminants (the “lights off” mode) [45]. When in contact with toxins, the luciferase activity of luminescent bacteria is directly inhibited, which reduces the related luminescence reactions (Figure 2) [46].

3.1. Vibrio fischeri in the Detection of Pollutants in Soil

Previous studies have used Vibrio fischeri for the acute biotoxicity testing of soil contaminated by heavy metals and organic pollutants [47,48,49,50,51,52]. Jiang Wei et al. used Vibrio fischeri to detect heavy metals (Ni2+, Pb2+, Cu2+, Cr2+, and Fe2+) in the soil of five chemical, metal, electrical, plastic, and coating plants in Xiamen [53]. L. Mariani et al. used Vibrio fischeri to detect the biotoxicity of organic contaminants (i.e., sodium laureth sulfate) in soil [54]. Yin Hongyang et al. used Vibrio fischeri to detect the joint toxicity of heavy metals (Cu and Cd) and organic pollutants (fenitrothion and ethyl ester) on soil, and the results showed that the biological toxicity of multiple pollutants was enhanced compared to a single pollutant [55]. Qiu Aifeng et al. used Vibrio fischeri to detect the combined pollution caused by Cu, Cd, and carbofuran in soil [56]. Cunha, Danieli et al. evaluated the sediment pollution biotoxicity caused by 39 pollutants (metals, polycyclic aromatic hydrocarbons, pesticides, and polychlorinated biphenyls) in the Itaipu–Piratininga lagoon system using Vibrio fischeri [57]. Giovanella, Patricia used Vibrio fischeri to detect the biotoxicity of soil contaminated by bioremediation diesel [58]. These applications show that Vibrio fischeri can be used to detect the biotoxicity of heavy metals, organic pollutants, and their combined contaminated soil.
Vibrio fischeri is a marine bacterium, and it needs NaCl (>3%) to glow (Table 1). However, such a high concentration of Na+ and Cl− affects the biological toxicity performance of some toxic substances, particularly heavy metals [59].

3.2. Photobacterium phosphoreum in the Detection of Pollutants in Soil

Photobacterium phosphoreum can be extracted and isolated from marine organisms such as corner shark whales [60]. Many studies have been conducted on toxicity detection using Photobacterium phosphoreum [61]. For example, Xu Hengpu et al. used Photobacterium phosphoreum T3 to measure the biological toxicity of Cu, Cd, and Pb individually and composite Cu-Cd and Cu-Pb in the contaminated soil [62]. Zeng Jianjun et al. used Photobacterium phosphoreum T3 to detect the biotoxicity of five heavy metals (Cu2+, Co2+, Zn2+, Fe3+, and Cr3+) and their binary mixtures [63]. Zhang Ying et al. used Photobacterium phosphoreum to detect the biological toxicity of soil polluted by environmentally persistent free radicals [64]. Zhao Jianguo et al. detected the toxicity of sludge caused by 4-chlorophenol using Photobacterium phosphoreum [65]. Shen Weihang et al. used Photobacterium phosphoreum to detect the biotoxicity of soil contaminated by artificial oil at different bioremediation stages [66]. Therefore, Photobacterium phosphoreum has good applicability in the biotoxicity detection of heavy metals, organic substances, and compound-contaminated soil.
Photobacterium phosphoreum is also a marine bacterium, and it needs NaCl (>3%) to glow (Table 1). Therefore, it is also affected by Na+ and Cl− during detection [67].

3.3. Vibrio qinghaiensis in the Detection of Pollutants in Soil

Vibrio qinghaiensis, sampled from Qinghai Lake by Chinese scientists in 1985, can emit light without Na+ [68]. It is a non-pathogenic freshwater luminescent bacterium and very sensitive to external toxic substances (e.g., phenolic compounds) [69,70,71]. Vibrio qinghaiensis has been used to test the acute biotoxicity of soil polluted by heavy metals and organic substances [72,73]. Chen Sumin et al. established a rapid detection method for soil biotoxicity polluted by Zn, Al, and Cu in a galvanized factory with Vibrio qinghaiensis sp.-Q67 as an indicator [74]. Liu Mingyuan et al. also used Vibrio qinghaiensis sp.-Q67 to detect the biological toxicity of soil-extracted pollutants (e.g., PAHs, alkyl PAHs, and phthalates) at different depths [75]. Han Dongmei used Vibrio qinghaiensis sp.-Q67 to measure the single and joint toxicity of Cd, Pb, and As in the soil [76]. Huang Ziyan et al. designed a ternary mixture system and used Vibrio qinghaiensis sp.-Q67 to explore the joint impact of mixed pollutants of heavy metal lead (Pb) and pesticides metalaxyl (MET) and glyphosate (GLY) on the environment [77].
In summary, the traditional luminescent bacteria bioassay can be used for the acute toxicity detection of soil contaminated by both organic and inorganic pollutants. However, there are some issues that still need to be addressed as follows: ① when luminescent bacteria are directly exposed to contaminated soil or a soil solution, soil particles absorb bacterial luminous signals; the turbidity and color of soil suspension also produce strong fluorescence interference. Therefore, the traditional luminescent bacteria bioassay is not applicable at present for directly detecting soil biological toxicity [78]. ② Some bioassays require pre-treatment of soil samples such as centrifugation, concussion, or leaching, which inevitably changes the physical and chemical properties of the pollutants. In addition, the reproducibility and sensitivity of the bioassays are poor [79,80].

4. Application of an Improved Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil

To overcome the above issues of the traditional luminescent bacteria bioassay, for example, the inability to achieve specific detection in the undisturbed soil, two novel methods have been developed as follows: (1) a luminescent bacteria method combined with gene recombination technology [81] and (2) luminescent bacteria functionalized with magnetic nanomaterials [82].

4.1. Recombinant Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil

In addition to detecting heavy metals, the traditional luminescent bacteria bioassay can also respond to thousands of pollutants and cannot achieve efficient identification of specific pollutants. Therefore, the recombinant luminescent bacteria bioassay has been developed with luminescent bacteria and genetic engineering, which has become a hot research topic in recent years [83,84].
The commonly used reporter genes in recombinant luminescent bacteria are firefly luciferase (luc), bacterial luciferase (lux), green fluorescent protein (gfp), enhanced green fluorescent protein (egfp), and cerulean fluorescent protein (cfp) [85]. Since the 1980s, researchers have successfully transcribed the luxCDABE gene box to allow it to be externally expressed in other host organisms (prokaryotic, eukaryotic, and plant cells). Among them, Escherichia coli is a traditional choice used for constructing host strains in recombinant luminescent bacteria due to its well-known genetic knowledge, complete cloning schemes, and rapid growth [86]. Therefore, recombinant luminescent bacteria using Escherichia coli as a host cell and the luminescent gene as a reporter gene can adapt to different environments and respond to different toxic substances, as well as conduct a sensitive, accurate, and comprehensive toxicity testing of pollutants, providing a basis for further pollution analysis and evaluation [87].
According to the expression mechanism of luminescent genes in recombinant luminescent bacteria, recombinant luminescent bacteria are usually divided into two types, i.e., constitutive and inducible types [88]. Constitutive recombinant luminescent bacteria usually use strong promoters to efficiently express reporter genes. Under toxic and harmful conditions, the expression level will decrease, and the reduced level is measurable and related to the toxicity of the sample. Therefore, in a constitutive expression system, any substance that reduces the cell growth rate or leads to cytotoxicity will result in a decrease in the reported signal intensity. These bacteria can detect total toxicity but cannot detect the toxicity of a specific pollutant. Therefore, although its luminous intensity is relatively stable, specific detection cannot be achieved [89]. The inducible recombinant luminescent bacteria are produced by introducing a recombinant plasmid carrying a reporter gene and a specific stress promoter into the host cell. Under normal conditions, the expression of luminescent reporter genes in these recombinant strains is inhibited or present at the lowest level, and the strains do not emit light or emit weak light. Only when a specific toxic substance is present is the promoter activated, and the luminescent reporter gene is transcribed and expressed to emit light [90]. The principle is that under normal conditions, regulator genes generate repressor proteins. The repressor protein binds to the operon manipulation gene, blocks the expression of structural genes, and inhibits transcription initiation. This causes the expression of luminescent genes to be inhibited, resulting in the strain not emitting light or emitting weak light. When specific pollutants exist, they bind to repressor proteins, causing conformational changes in repressor proteins that cannot bind to manipulating genes. Therefore, RNA polymerase can normally catalyze the transcription of structural genes on the operon, that is, the operon is induced to express and start transcription. The luminescent gene was expressed, and the strain was induced to emit light by this toxicity [91]. Due to the specific toxicity recognition characteristics of inducible recombinant luminescent bacteria, most of the constructed bacteria at home and abroad are inducible recombinant bacteria [92].
Some scholars have applied constitutive and inducible recombinant luminescent bacteria to detect the acute toxicity of soil pollution. Fang Guizhen et al. selected genetic recombination luminescent bacteria E. coli HB101 pUCD607 and Vibrio fischeri, to, respectively, study the biotoxicity of soil pollution with eight heavy metals (Zn2+, Cu2+, Fe3+, Hg2+, Mn2+, Cd2+, Co2+, and Cr6+) [93]. The results showed that the half effect mass concentration (EC50) of genetic recombinant luminescent bacteria E. coli HB101 pUCD607 was higher than Vibrio fischeri, which indicated that the recombinant luminescent bacteria were more sensitive than Vibrio fischeri in detecting metal toxicity in the soil. Wang Yongzhi et al. constructed luminescent bacteria with mercury-sensitive and anti-mercury genes to detect the toxicity of mercury ions, and the results showed that the recombinant strains had high sensitivity and specificity for detecting soil pollution with mercury ions [94]. He Wei et al. introduced fusion fragments (mer-egfp and nah-luxCDABE) carrying regulatory gene elements and reporter genes into host cells to construct recombinant luminescent bacteria. Two sensor strains have been shown to produce significant fluorescence or bioluminescence in the presence of mercury and phenanthrene (PHE). In soil test samples, the detected mercury threshold and PHE threshold range from approximately 19.6 to 111.6 and 21.5 to 110.9 mg kg−1. By using this method of constructing recombinant luminescent bacteria to detect mercury and PHE in soil, the detection range obtained is higher than the detection limit obtained using traditional analytical methods, indicating that the recombinant luminescent bacteria method has higher sensitivity. Therefore, the two recombinant luminescent bacteria constructed by He Wei and others can quickly, sensitively, and specifically detect mercury- and PHE-contaminated soil [95]. Rushika Patel et al. fused two mono- and poly-aromatic hydrocarbon promoters with the reporter genes gfp and cfp, respectively, to produce two recombinant luminescent bacteria. In terms of specificity detection, these two recombinant luminescent bacteria are very effective for the detection of aromatic hydrocarbons. Additionally, the induction time is shorter (30 min), and detectable as low as a 0.1–1 μm substrate. The results show that the detection efficiency of the recombinant luminescent bacteria constructed by Rushika Patel et al. for aromatic pollutants in environmental and industrial samples is satisfactory and reliable [96].
In summary, the recombinant luminescent bacteria method can improve the sensitivity and specificity in the toxicity detection process and has good practicality in the detection of soil pollution caused by heavy metals and organic pollutants.

4.2. Application of MNP-Based Biosensors in Soil Detection

The recombinant luminescent bacteria bioassay can realize specific detection of heavy-metal-polluted soil and enhance the sensitivity of luminescent bacteria. However, this bioassay still cannot realize the detection of biological toxicity of undisturbed soil. However, MNP-based biosensors are derived from a new interdisciplinary frontier associated with chemistry, biotechnology, and material science, and can be used to detect pollutants in undisturbed soils [97].
Biosensors are devices that detect the presence of physical/biochemical changes in substrates using biological components and provide quantifiable signals [98]. The substance to be measured enters the biologically active material through diffusion and undergoes molecular recognition and a biological reaction. The generated information, such as chemical changes, thermal changes, and light changes, is then converted into quantifiable and processable electrical signals by corresponding physical or chemical transducers, which are amplified and output by a secondary instrument to detect the concentration of the substance. Biosensors are composed of two main key components: a molecular recognition component, which is a biosensor signal-receiving or -generating component, and a physical signal conversion component, which is a hardware instrument component [99]. Due to its high sensitivity, low limit of detection (LOD), rapidity, and cost-effectiveness, the application of biosensors in environmental detection is booming [100]. Magnetic nanomaterials refer to magnetic metal or metal oxide materials with a size in the range of 1–100 nm, which is an important component of nanomaterials. Due to their unique magnetic and nanomaterial properties, magnetic nanoparticles are widely studied and applied in many fields. MNPs are a special class of magnetic nanomaterials, which are more widely used. Currently, magnetic nanoparticle materials mainly include metal oxides and complexes such as Fe, Mn, and Co, among which materials containing Fe are the most common. The Fe3O4 magnetic nanoparticle has become one of the most widely used magnetic nanoparticles due to its simple preparation, high stability, low cost, good biocompatibility, good magnetic responsiveness, and easy surface modification [101].
Therefore, based on the excellent performance of biosensors and MNPs, they can be assembled into MNP-based biosensors as MNPs have positive surface charges, and the electrostatic interaction between MNPs and microbial cells combines them together and endows them with magnetic properties [102]. Thus, MNP-based biosensors can be separated under the action of an external magnetic field after they react with the samples in the environment.
The samples are then put in a sodium chloride solution to determine their biological toxicity using a biotoxicity detector (Figure 3). In this way, the detection of pollutants in undisturbed soil is realized [103].
Some scholars have studied the use of MNPs to functionalize bacteria and thus construct new multifunctional biosensors, and they used it to detect the biological toxicity of pollutants [104,105]. For example, Zhu Nali et al. used recombinant bacteria and MNP modified with silica and polyvinylimine to construct assemblies for detecting heavy metal pollution [106]. D. Saed et al. assembled MFe3O4 nanoparticle construction assembles on the cell wall of Gram + ve Micrococcus lutes RM1. This assembly had a good adsorption and degradation capacity for Polyaromatic hydrocarbons (PAHs) pollutant pyrene [107]. Cai Yahui et al. prepared magnetic bacteria–polymer complexes to efficiently detect and remove the phenol and Dimethylformamide [108]. Jia Jianli et al. used a magnetic nanomaterial functionalized whole-cell biosensor to detect the biological toxicity of soil contaminated by heavy metal chromium in a cinder yard. The influence of soil particles was avoided during detection [109]. Zhang Kai et al. used MNPs and luminescent bacteria to construct an MNP-P. phosphoreum biosensor and detected the biotoxicity of soil contaminated with heavy metals, and the sensor realized the undisturbed detection of contaminated soil [110].
To summarize, using magnetic nanoparticles and bacteria to assemble sensors can help detect the biological toxicity of contaminated soil caused by heavy metals and organic substances. In a sense, it can solve the lack of nondestructive testing of the biological toxicity of contaminated soil.

5. Conclusions and Prospects

This paper summarizes the application, luminescence mechanism, and soil pollution detection mechanism of three commonly used luminescent bacteria: Vibrio fischeri, Photobacterium phosphoreum, and Vibrio qinghaiensis. Methods for improving the sensitivity and specificity of detection were also proposed by gene recombination combined with luminescent bacteria, which are functionalized with magnetic nanomaterials. Although the two new methods have some advantages over traditional ones, there are still some problems to be solved. The method using recombinant luminescent bacteria cannot detect pollution in undisturbed contaminated soil. The reproducibility of a magnetic nanobacterial sensor is poor, and the negative magnetic effect between magnetic nanoparticles and luminescent bacteria is not clear. Therefore, it is also necessary to improve the performance of the magnetic nanobacteria sensor and make it more applicable to the detection of undisturbed soil. However, considering the current issue, that toxicity testing of contaminated soil cannot achieve undisturbed soil detection, the method of combining MNPs with luminescent bacteria to prepare sensors can effectively solve this problem. This method separates the sensor from the contaminated soil through the action of an external magnetic field, without the need for extraction and other operations. It achieves the detection of undisturbed contaminated soil, which has good application prospects. In the future, further research on its application and promotion mechanism can be carried out to achieve a wider application of this sensor in the detection of contaminated soil.

Author Contributions

Conceptualization, K.Z. and M.L.; methodology, K.Z. and M.L.; software, M.L.; validation, K.Z. and M.L.; formal analysis, M.L. and X.S.; investigation, X.S. and D.W.; resources, K.Z. and M.L.; data curation, K.Z. and M.L.; writing—original draft preparation, K.Z. and M.L.; writing—review and editing, K.Z. and M.L.; visualization, D.W.; supervision, X.S.; project administration, K.Z.; funding acquisition, K.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (42177037) and the Key Research and Development Program of Autonomous Region (2022B03028-1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We acknowledge all the authors for their contributions. We sincerely thank the anonymous reviewers and the editor for their effort in reviewing this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Mechanism diagram of chemical reactions and luminescence in luminescent bacteria under the influence of fluorescein gene.
Figure 1. Mechanism diagram of chemical reactions and luminescence in luminescent bacteria under the influence of fluorescein gene.
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Figure 2. The biotoxicity of pollutants was measured by changes in the luminous intensity of luminescent bacteria. The decrease in luminous intensity is due to the interaction of the luminescent bacteria with the pollutants, which inhibits the production of luciferase by suppressing transcription and translation. This leads to the inhibition of luminescence reaction based on bacterial luciferase mediation, resulting in a decrease in luminous intensity.
Figure 2. The biotoxicity of pollutants was measured by changes in the luminous intensity of luminescent bacteria. The decrease in luminous intensity is due to the interaction of the luminescent bacteria with the pollutants, which inhibits the production of luciferase by suppressing transcription and translation. This leads to the inhibition of luminescence reaction based on bacterial luciferase mediation, resulting in a decrease in luminous intensity.
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Figure 3. Step diagram for biotoxicity detection of soil pollution by MNP-based biosensors. The sensor is first placed in a soil dilution solution and separated by an plus magnetic field after the action. The sensor is then placed in NaCl solution and finally the change in luminous intensity is measured using an intelligent biological toxicity tester to determine the biotoxicity.
Figure 3. Step diagram for biotoxicity detection of soil pollution by MNP-based biosensors. The sensor is first placed in a soil dilution solution and separated by an plus magnetic field after the action. The sensor is then placed in NaCl solution and finally the change in luminous intensity is measured using an intelligent biological toxicity tester to determine the biotoxicity.
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Table
Table 1. Comparison of conditions of the three luminescent bacteria used in traditional luminescent bacteria bioassay used to detect pollutants in the environment.
Table 1. Comparison of conditions of the three luminescent bacteria used in traditional luminescent bacteria bioassay used to detect pollutants in the environment.
Luminescent BacteriaSourceSalinity EffectTemperaturepHResuscitation FluidToxicity ControlReferences
Vibrio fischeriOceanYes18–30 °C6.5–7.53% NaCl solutionHgCl2[39]
Photobacterium phosphoreumOceanYes20–25 °C6.5–7.53% NaCl solutionHgCl2[39,40]
Vibrio qinghaiensisFresh waterNo18–30 °C6.0–9.00.85% NaCl solutionPhenol[39,40,41]
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Zhang, K.; Liu, M.; Song, X.; Wang, D. Application of Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil. Sustainability 2023, 15, 7351. https://doi.org/10.3390/su15097351

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Zhang K, Liu M, Song X, Wang D. Application of Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil. Sustainability. 2023; 15(9):7351. https://doi.org/10.3390/su15097351

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Zhang, Kai, Meng Liu, Xinlong Song, and Dongyu Wang. 2023. "Application of Luminescent Bacteria Bioassay in the Detection of Pollutants in Soil" Sustainability 15, no. 9: 7351. https://doi.org/10.3390/su15097351

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