Microbial Biosensor for Marine Environments

Abstract

A biosensor is an analytical device which integrates a biological recognition element with a physical transducer to generate a measurable signal proportional to the concentration of the analyses. A microbial biosensor consists of a transducer in conjunction with immobilized viable or non-viable microbial cells. Non-viable cells obtained after Permeable or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Based on the sensing technique, recent reported microbial biosensors can be classified into two major groups: electrochemical microbial biosensors and optical microbial biosensors. Microbial biosensors based on light emission from luminescent bacteria are being applied as a sensitive, rapid and non-invasive assay in several biological systems. Bioluminescent bacteria are found in nature, their habitat ranging from marine (Vibrio fischeri) to terrestrial (Photorhabdus luminescens) environments. Bioluminescent whole cell biosensors have also been developed using genetically engineered microorganisms (GEM) for the monitoring of organic, pesticide and heavy metal contamination. The major application of microbial biosensors is in the environmental field. Currently, different microorganisms have been genetically engineered to respond to particular stresses or toxicity and are described in the literatures as biosensors for environmental stressesthough for them to be biosensors in these of this publication these organisms would have to be coupled to a suitable transducer. Based on the respiration of cells, Pseudomonas sp. entrapped polycarbonate membrane modified oxygen electrode was also applied to detect PNP which serves as the sole carbon and nitrogen source for the bacteria. By application of these biosensor in the marine environment the pollution in these ecosystems can be better manage.

Full text

BEPLS Vol 3 [Spl Issue V] 2014 1 | P a g e ©2014 AELS, INDIA
Bulletin of Environment, Pharmacology and Life Sciences
Bull. Env. Pharmacol. Life Sci., Vol 3 [Special Issue V] 2014: 01-13
©2014 Academy for Environment and Life Sciences, India
Online ISSN 2277-1808
Journal’s URL:http://www.bepls.com
CODEN: BEPLAD
Global Impact Factor 0.533
Universal Impact Factor 0.9804 REVIEW ARTICLE
Microbial Biosensor for Marine Environments
Pariya Ahmadi Balootaki1, Mehdi Hassanshahian2*
1. Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran.
2. Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran.
Email: mshahi@uk.ac.ir
ABSTRACT
A biosensor is an analytical device which integrates a biological recognition element with a physical transducer to
generate a measurable signal proportional to the concentration of the analyses. A microbial biosensor consists of a
transducer in conjunction with immobilized viable or non-viable microbial cells. Non-viable cells obtained after
Permeable or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for
enzymes. Based on the sensing technique, recent reported microbial biosensors can be classified into two major groups:
electrochemical microbial biosensors and optical microbial biosensors. Microbial biosensors based on light emission
from luminescent bacteria are being applied as a sensitive, rapid and non-invasive assay in several biological systems.
Bioluminescent bacteria are found in nature, their habitat ranging from marine (Vibrio fischeri) to terrestrial
(Photorhabdus luminescens) environments. Bioluminescent whole cell biosensors have also been developed using
genetically engineered microorganisms (GEM) for the monitoring of organic, pesticide and heavy metal contamination.
The major application of microbial biosensors is in the environmental field. Currently, different microorganisms have
been genetically engineered to respond to particular stresses or toxicity and are described in the literatures as biosensors
for environmental stressesthough for them to be biosensors in these of this publication these organisms would have to be
coupled to a suitable transducer. Based on the respiration of cells, Pseudomonas sp. entrapped polycarbonate membrane
modified oxygen electrode was also applied to detect PNP which serves as the sole carbon and nitrogen source for the
bacteria. By application of these biosensor in the marine environment the pollution in these ecosystems can be better
manage.
Key words: Bacteria, Biosensor, Contamination, Environment, Ecology, Detection.
Received 10.05.2014 Revised 19.06.2014 Accepted 25.09. 2014
INTRODUCTION
Biosensors have been under development for over 35 years and research in this field has become very
popular for 15 years. Electrochemical biosensors are the oldest of the breed, yet sensors for only one
analyze (glucose) have achieved widespread commercial success at the retail level (1).What is a
biosensor? In the early days (the 1960s and1970s), a sensor seemed to always be a probe of some sort,
perhaps due to a vision inextricably linked to pH, ion selective or oxygen electrodes. If you follow the old
literature, you will find biosensors that were called bio electrodes or enzyme electrodes or bio catalytic
membrane electrodes (2). More recently, we have seen the definition broadened to include sensors
buried within large automate instruments (3). There are some who see mass spectrometries,
chromatography or electrophoresis as a viable sensor component (4).Many sensors used for biological
purposes are therefore not biosensors, including those for temperature, pressure, electrocardiograms,
pH, Ca2+, catecholamines and the like. By contrast, it is fair to consider surface Plasmon resonance (SPR)
devices as utilizing biosensors (3, 5). Even labeled nanoparticles imbedded in the cytosol of individual
cells that report optically are viable sensors.
Main description of biosensors
A biosensor is an analytical device which integrates a biological recognition element with a physical
transducer to generate a measurable signal proportional to the concentration of the analyses (6, 7, 8, 9,
10, 11, 12). In the general scheme of a biosensor, the biological recognition element responds to the
target compound and the transducer converts the biological response to a detectable signal, which can be

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measured electrochemically, optically, acoustically, mechanically, calorimetrically, or electronically, and
then correlated with the analyze concentration (7, 8, 12, 13). Since Clark and Lyon developed the first
biosensor for glucose detection in 1962, biosensors have been intensively studied and extensively utilized
in various applications, ranging from public health and environmental monitoring to homeland security
and food safety (1, 11, 14, 15, 16).Various biological recognition elements, including cofactors, enzymes,
antibodies, microorganisms, organelles, tissues, and cells from higher organisms, have been used in the
fabrication of biosensors (9). Among these biological elements, enzymes are the most widely used
recognition element due to their unique specificity and sensitivity (17). However, the purification of
enzyme is costly and time-consuming. In addition, the in vitro operating environment could result in a
decrease of the enzyme activity (13). Microbes (e.g., algae, bacteria, and yeast) offer an alternative in the
fabrication of biosensors because they can be massively produced through cell culturing. Also, compared
to other cells from higher organism's such as plants, animals, and human beings, microbial cells are easier
to be manipulated and have better viability and stability in vitro (13), which can greatly simplify the
fabrication process and enhance the performance of biosensors.
Microalgae usually proposed as bio receptors in sensors for water toxicity testing are not simple to
handle and need to be appropriately sampled, diluted and immobilized on Alteration membranes before
use. The species employed in this study, being a colonial microalga forming macroscopic mats, does not
require any immobilization procedure, thus making easier and faster the preparation of each analysis.
The proposed biosensor was quite sensitive toatrazine and showed intermediate sensitivity to carbonyl,
while the detection of toxicity in the case of heavy metals was slow. This could be due either to biological
factors (related to the adsorption characteristics of the cell walls as well as to specific pathways for
sequestration, metabolization and release of the algal species used) or to chemical reasons such as the
presence of natural completing agents and the high pH of the experimental medium. Another
circumstance that may have resulted in decreased metal toxicity is the production of extra-cellular
ligands by the alga, a pattern displayed by many cyanobacteria in response to metal stress (18, 19). Figure
(1) shows the schematic representation of a biosensor.
Figure 1. The schematic representation of a biosensor
What is microbial biosensor?
A microbial biosensor consists of a transducer in conjunction with immobilized viable or non-viable
microbial cells. Non-viable cells obtained after Permeable or whole cells containing periplasmic enzymes
have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory
and metabolic functions of the cell, the analyze to be monitored being either a substrate or an inhibitor of
these processes. Bioluminescence-based microbial biosensors have also been developed using genetically
engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for
toxicity and bioavailability testing. Figure (2) show the schematic representation of a microbial biosensor
(20).
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Figure 2: The schematic representation of a microbial biosensor
Classification of biosensors:
Based on the sensing technique, recent reported microbial biosensors can be classified into two major
groups: electrochemical microbial biosensors and optical microbial biosensors. Amperometry is the most
widely used technique in electrochemical microbial biosensors. Amperometric microbial biosensors have
been extensively exploited for environmental applications (17, 21, 22). However, this method is time
consuming and not suitable for on-line monitoring (23). Other electrochemical microbial biosensors: The
other recently published electrochemical microbial biosensors based on conductometric, potentiometric,
voltammetric transducers as well as MFC-based biosensors.
The conductometric microbial biosensor is attractive and appealing owing to its fast and sensitive
response to the analyses. In this regard, a conductometric biosensor using C. vulgaris microalgaeas the
bioreceptor was constructed to detect heavy metal ions and pesticides in water sample (24).
Potentiometric microbial biosensors detect the amount of analyses by measuring the potential difference
between the working electrode and the reference electrode separated by a selective membrane. Recently,
a potentiometric biosensor based on the pH electrode modified by permeable P. aeruginosa was
developed for selective and rapid detection of cephalosporin group of antibiotics (25). The hydrolysis of
cephalosporin, due to the enzyme activity of the microbial layer, was accompanied by the production of
protons near the pH electrode. The response came from the change of electric potential difference
between the working electrode and the reference electrode. Another potentiometric biosensor for the
identification of Beta-lactam residues in milk was also reported (26). These biosensors classified as
follow:
A) Fluorescent microbial biosensors:
Based on the detection mode, fluorescent microbial biosensors can be divided into two categories: in vivo
and in vitro. In vivo fluorescent microbial biosensor makes use of genetically engineered microorganisms
with transcriptional fusion between an inducible promoter and a reporter gene encoding fluorescent
protein. Green fluorescent protein (GFP), encoded by gfp gene, is among the most popular tools due to its
attractive stability and sensitivity, and the fluorescence emitted by GFP can be conveniently detected by
modern optical equipments with little or no damage to the host system(27).
B) Bioluminescent microbial biosensors:
A bioluminescent microbial biosensor measures the luminescence change emitted by living
microorganisms. The luminescence change is, in fact, caused by lux gene-coded luciferase responding to
the target analyze in a dose-dependent manner. The expression of lux gene in microorganisms can be
controlled in either a constitutive or inducible manner. In the constitutive manner, the lux gene exists
constitutively in the sensing microbe and the bioluminescence will change directly with the addition of
chemicals of interest. Based on the act that the light intensity produced by the bacteria could be reduced
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in the presence of toxic compounds, a Vibrio fischeri based bioluminescent microbial biosensor was
developed for the rapid determination of the toxicity of some common environmental pollutants in a
continuous flow system (28).A tetracycline (TC) luminescent whole-cell biosensor was developed for the
rapid and specific TC residue assay in poultry muscle tissue with membrane permeabilizing agent
polymyxin Band sensitizing agent EDTA (29).
C) Sodium channel-based biosensors:
The effect of PSP toxins (STX, gonyautoxin and tetrodotoxin) as sodium channel blockers has been
exploited for the development of a tissue biosensor (30, 31). The authors covered a Na electrode with a
frog bladder membrane, rich in sodium channels, and integrated into a flow cell. Investigating the
transport of Na+ ions, they could detect the toxin presence. The toxicity levels of the toxins correlated
with those determined with the mouse bioassay and, in the tetrodotoxin case, the biosensor was able to
detect concentrations more than one order of magnitude below the limit of the detection of the bioassay.
A particular case is the neuronal network biosensor developed by Kulagina et al. (32) which exploits the
effect of STX and PbTx-3 on the extracellular action potentials. The biosensor was constructed by growing
cultured mammalian neurons from spinal cord tissue of embryonic mice over a 64-site microelectrode
array. Despite the distinct actions of these two toxins on the nervous tissue (STX inhibits propagation of
action potentials and PbTx-3 enhances activation of thesodium channels), both inhibited mean spike rate
of spinal cord neuronal networks. The detection limits for STX and PbTx-3were, respectively, 12 and 296
pgmL−1 in buffer and 28 and430 pgmL−1 in 25-fold-diluted seawater. These extremely low values
(approximately 30 000 times below the mouse bioassay detection limit for STX and 300 times below the
regulatory limit for PbTx-3) are due to the extremely high sensitivity of the spinal cord networks.
Additionally, the array responded to the presence of toxin-producing algae but not to the presence of non-
toxin isolates of the same algal genera. Although this generic approach cannot fully identify or quantify
individual toxins, its application as screening tool is clearly justified.
D) Enzyme inhibition-based biosensors:
Hamada-Sato et al have recently developed a biosensor that combines the PP2A inhibition with the
phosphate ion consumption by pyruvate oxidase (PyOx) into a flow injection analysis (FIA) system (33).
However, the inhibition step is performed in a microtube and only the second enzyme is immobilized.
Nevertheless, they measured OA with a detection limit of 0.1 ngmL−1, the biosensor being 50 times more
sensitive than ELISA.
Our group is currently working on the development of an electrochemical PP2A inhibition-based
biosensor for the determination of OA. Colorimetric experiments with the enzyme immobilized by
entrapment with poly (vinyl alcohol) azide-unitpendant water-soluble photopolymer (PVA-AWP) on
screen printed carbon electrodes have demonstrated the viability of the approach and its applicability to
the detection of the toxin in mussels. Our strategy is much simpler than the one mentioned above, since
the enzyme inhibition is detected directly using appropriate PP2A substrates, electrochemically active
only after the dephosphorylation by the enzyme (33).
USE OF MICROBIAL CELLS AS BIOSENSING ELEMENTS
Microbes have a number of advantages as biological sensing materials in the fabrication of biosensors.
They are present ubiquitously and are able to metabolize a wide range of chemical compounds.
Microorganisms have a great capacity to adapt to adverse conditions and to develop the ability to degrade
new molecules with time. Microbes are also amenable for genetic modifications through mutation or
through recombinant DNA technology and serve as an economical source of intracellular enzymes. (34,
35)
Selection of an appropriate culture is essential as the specific microbial species used in biosensors have
characteristic substrate spectra which may or may not correspond well with the spectrum of compounds
present in the sample. Adaptation of a microbe for induction of desirable metabolic pathways and uptake
systems by cultivation in medium containing appropriate substrates may often be desirable (36, 37, 38).
Microbial biosensors based on light emission from luminescent bacteria are being applied as a sensitive,
rapid and non-invasive assay in several biological systems (39) (40). Bioluminescent bacteria are found in
nature, their habitat ranging from marine (Vibrio fischeri) to terrestrial (Photorhabdus luminescens)
environments. Bioluminescent whole cell biosensors have also been developed using genetically
engineered microorganisms (GEM) for the monitoring of organic, pesticide and heavy metal
contamination. The microorganisms used in these biosensors are typically produced with a constructed
plasmid in which genes that code for luciferase are placed under the control of a promoter that recognizes
the analyze of interest. When such microbes metabolize the organic pollutants, the genetic control
mechanism also turns on the synthesis of luciferase, which produces light that can be detected by
luminometers.(41)
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Microbial biosensors for environmental applications
The major application of microbial biosensors is in the environmental field (42, 43, 44, 45, 46, 47, 48, 49,
50, 51). Microbial biosensors have been developed for assaying BOD, a value related to total content of
organic materials in wastewater. BOD sensors take advantage of the high reaction rates of
microorganisms interfaced to electrodes to measure the oxygen depletion rates.
A microbial biosensor consisting of an oxygen microelectrode with microbial cells immobilized in
polyvinyl alcohol has been fabricated for the measurement of bio available organic carbon in toxic
sediments. The biosensor allows the estimation of available dissolved organic carbon in sediment profiles
on a micro scale (52). Optical fiber (53) and calorimetric (54) based transducers have been used in BOD
biosensors.
Halogenated hydrocarbons used as pesticides, foaming agents, flame-retardants, pharmaceuticals and
intermediates in the polymer production are one of the largest group of environmental pollutants.
Microbial bio assays using immobilized cells of Rhodococcus strain containing alkyl-halide hydrolase has
been described by Hutteret al. (55). The enzyme presents in the cell liberate shalogen ions from
halogenated hydrocarbons. These studies were extended in the fabrication of a microbial sensor (56). The
sensor can be stored in the dry form at 277 K for 1 week. More recently a gram-positive actinomycete like
organism, exhibiting a broad spectrum for the dehalogenation of halogenated hydrocarbons, has shown
better promise and may have potential in the fabrication of a broad specificity biosensor for halogenated
hydrocarbons (57).
Applications of microbial biosensors in food, fermentation and allied fields
In recent years, the demand for quick and specific analytical tools for food and fermentation analysis has
increased and is still expanding. Both industry and government health agencies require a wide array of
different analytical methods in the quality assurance of food materials. Analysis is needed for monitoring
nutritional parameters, food additives, food contaminants, microbial counts, shelf life assessment and
other factory characteristics like smell and odor. A variety of sensors based on enzymes and antibodies
(58, 59, 60, 61) as wells electronic noses (62, 63) have been reported. Microbial biosensors have also
shown potential in food analysis (60). A number of reports are available on microbial biosensors for
amino acids such as tyrosine (Aeromonasphenologenes), tryptophan (Ps. fluorescens) and glutamic acid (B.
subtilis). (64) (65). Determination of phenylalanine is needed not only forthe process control of the
phenylalanine fermentation but also for the neonatal diagnosis and dietary management of hyper
phenylalaninaemia. A microbial biosensor based on Proteus wulgaris cells immobilized in Ca-alginate on
an amperometric oxygen electrode has been reported (66). Phenylalanine deaminase present in the cell
oxidises phenylalanine to phenylacetic acid.
The importance of microbial biosensors in the marine environments:
Recently appeared around shellfish (diarrheic, paralytic, amnesic, neurologic and azaspiracid) and fish
(ciguatera and puffer) poisonings produced by different types of phycotoxins, making evident the urgent
necessity of counting on appropriate detection technologies. With this purpose, several analysis methods
(bioassays, chromatographic techniques, immune assays and enzyme inhibition-based assays) have been
developed. However, easy-to-use, fast and low-cost devices, able to deal with complicated matrices, are
still required. Biosensors offer themselves as promising bio tools, alternative and/or complementary to
conventional analysis techniques, for fast, simple, cheap and reliable toxicity screening.
A whole-cell sensor system that has been applied to the analysis of seawater utilizes the marine algae
Spirulina subsalsa coupled to a Clark-type oxygen electrode in a flow through system to estimate pollution
as indicated by variation in photosynthetic activity (67). Substances tested included chlorophenols,
pesticides and surfactants. A similar system based on C. vulgaris and fiber optic signal detection was
described for atrazine, simazine, isoproturon and diuron, with sub-ppb detection limits for photosystemII
inhibitors (68). The advantage of whole cell sensors in this context is that they give information
concerning bioavailability and potentially measure physiological responses, which are relevant to marine
processes. The disadvantages that the obtained signals are generally less specific than those collected
with enzymatic or affinity sensors and might have to be backed up by chemical analysis to resolve
causative relationships between contaminants.
A topic specific to marine monitoring is the pollutants introduced to the marine environment through
produced water and drilling fluids from oil exploration platforms (69, 70, 71). Produced water can
contain a cocktail of ingredients from hydrocarbons to antifoam agents, biocides, surfactants, corrosion
inhibitors and emulsifiers, which can have acute or chronic toxic effects. Given the large range of
chemicals potentially implicated, it is likely that an estimate of toxicity/risk posed using a complex and
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preferably relevant biological system would be a useful tool in impact assessment. Currently, different
microorganisms have been genetically engineered to respond to particular stresses or toxicity and are
described in the literatures “biosensors for environmental stresses” (72, 73, 74, 75), though for them to
be biosensors in these of this publication these organisms would have to be coupled to a suitable
transducer.
Another typically marine problem is the widespread contamination of water and sediments with
antifouling agents, most notoriously in compounds such as tributyltin (TBT). Despite the fact that an
outright ban agreed by the International Maritime Organization (IMO) is currently being implemented,
TBT is still prevalent in many sediments and it quantification and removal is a problem that will face
marine scientists and regulators for years to come (76). Among the best-documented effects of organotins
on biota are direct toxicity, shell thickening in oysters, a decline in recruitment of their juvenile stages,
and endocrine disruption. Given that TBT has been found to yield effects at concentrations in the low ng/l
range (77), any detection methods would have to be extremely sensitive. A method based on a bacterial
bioluminescence based bioassay for the specific detection of organotin compounds was reported (78).
The detection limits were found to be 0.08 _M for TBT (26_g/l) and0.0001_M for DBT (0.03_g/l) with a
linear range of one logarithm. The application of the bioassay to environmental samples is still under
development and will depend on the contamination levels in relation to the detection limit of the bioassay
for TBT and DBT. A flow-through sensor based on this assay with the bacteria immobilized within a chip
and luminescent detection has recently been presented at the Eighth World Congress on Biosensors (79).
Accumulation of the organotin within the immobilization matrixes assumed to be responsible for the
improved detection limit of 1 nM TBT (325 ng/l).
Trace metals
Trace metals in the marine environment can have dual roles: in some cases they can act as essential
limiting trace elements such as, for example, iron which has been found to limit algae growth in some
parts of the ocean (80, 81, 82), in other cases they can present a pollution issue. A number of biosensors
for metals have been described in the literature such as are combining luminescent bacterial sensors for
the detection of zinc and chromate (83), a whole-cell sensor using the alkaline phosphates activity of the
algae C.vulgaris and inter digitized conductometric electrodes for the detection of cadmium ions (84), an
amperometric detection system based on urease inactivation for the screening of heavy metals such as
mercury, copper, cadmium and tin in soil leachates (85), a catalytic cDNA sensor with fluorescent
detection for lead (86) and many more. One factor of great importance in determining the fate and effect
of metals in the environments their bioavailability and this is an issue that can, in certain instances, be
addressed by the use of biosensors rather than chemical analysis in their detection. Two different sensor
systems, one whole-cell and one protein-based, for metal bioavailability have been described (87) and
some of the issues underlying the use of biosensors in assessing metal bioavailability have recently been
discussed (88). Since the analysis and speciation of trace metals is a vast field in marine chemistry, the
use of biosensors to obtain additional information appears a sensible route forward.
Food safety
Pollution of seafood through algal toxins is a concern that requires a comprehensive monitoring
programmed. Paralytic shell fish poisons (saxitoxin, gonyautoxin and tetrodotoxin) act as sodium channel
blockers. A tissue biosensor based on frog-bladder membrane and a sodium electrode has been
developed for the sensitive detection of these toxins (89, 90). An electrochemical immune sensor
forsaxitoxin and brevetoxin using glucose oxides as enzyme label has been described (91), as well as one
for okadaic acid, brevetoxin, domoic acid and tetrodotoxin, based on a screen-printed electrode (SPE)
system and alkaline phosphatase as enzymatic label (92). Okadaic acid, a diarrheic shellfish poison, was
detected by chemi luminescent immune sensor in mussel homogenate (93) and also using a quartz crystal
micro balances transducer for the immunoreaction (94).
The biosensors can have a range of applications in marine science, some of which will complement
chemical methods in providing information about interactions with biological material; some will have
advantages in terms of field-applicability, automation or cost.
What kinds of biosensors have been used for witch material?
When the Nernstian biosensor response was used for calibration, up to 20,000 mgL−1 glucose standard
was measured without sample dilution. BOD calibrations were accomplished using the two more
commonly used standard artificial wastewaters, GGA and OECD solutions. The results showed that the
potentiometric CO2 electrode was a useful transducer, allowing us to build, calibrate and characterize a
BOD-like biosensor. Moreover, limitations present at oxygen amperometric electrode (customarily used
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as BOD biosensor-based transducer) such as oxygen low solubility and its reduction at the cathode were
avoided.
Fluorescence can be detected at a longer wavelength after the excitation of the fluorescent substance at a
shorter wavelength. Fluorescent biosensors have been widely applied in analytical chemistry due to their
easy construction using standard molecular biology techniques (95). Fluorescence-based microbial
biosensors can be divided into in vivo and in vitro type. For the in vivo type fluorescent microbial
biosensor, the microorganisms are able to produce the fluorescent substance (e.g., green fluorescent
protein) without the addition of an exogenous fluorescent element. For the in vitro type, the metabolic
activity of microorganisms changes the environment surrounding them, resulting in the change of light
emission due to the exogenous fluorescent element.
Based on the respiratory activity of the bacteria, target analyze adapted P. putida has been used as the
sensing element in the detection of other pollutants, such as BTE (benzene, toluene, ethylbenzene) (96)
and 2,4-dichloro phenoxy acetic acid (97).
Candida tropicalis (98) have been applied to the construction of ethanol whole-cell biosensors. Moreover,
a G. oxidants-based amperometric biosensor with ferric cyanide serving as the mediator for the
measurement of ethanol in FIA system was constructed (99).
Marine microalgae, especially phycotoxin producers such as several species of dinoflagellates and
diatoms, are one of the main problems in the exploitation of marine resources around the world.
Phycotoxins are toxic compounds that enter into the food chain as components of the phytoplankton.
Shellfish ingest these toxins and act as vectors, transmitting them to humans; and not only shellfish:
several marine carnivores, such as some fish species and crabs, may also act as vectors. Phycotoxins
accumulate in the digestive glands of shell fish without causing any toxic effect on it. However, when
Material
Kind
of bacteria
Substrate
Reference
Naphthalene
Sphingomonas Sp
. Or
P.Fluorescens
Hydrogel
(135, 136)
Acrylamide/Acrylic Acid/Acrylonitrile
Brevibacterium Sp
.
Waste Waters
(137)
Acrylonitrile
P. Pseudoalcaligenes
Waste Waters
(137, 138)
Cyanide
S.
cerevisiae
Cyanide
(139)
Nitrification
A Microbial Sensor
Waste Waters
(140)
Determination Of Phytotoxicity
Synechococcus Sp.
Isolated Chloroplasts Or
Photosynthetic Membranes
(141)
Detecting Pollutant
-
Induced Effects
Cyanobacterium
Photoelectrochemical Cell
(141)
Sulphite
Thiobacillus thiooxidans
Foods
(142, 143)
Pharmaceutical
B. Subitilits
Enalaprilmaleate (Ema)
(38)
Highly Toxin Materials, Including
Heavy Metals And Organic Chemicals
Photobacterium
phosphoreum
Based On Heat
Shock Gene
-
Bioluminescence
(144)
(145)
(146)
(147)
Biocides
E. coli
Biocides
(148)
GEM
Biolumine Scene (
E.
coli)
Environment
(149)
Cytotoxicity And Lysis
S. cerevusiae
Sense Chemicals
(150)
For The Measurement Of Sugars
Dienococcus
Food And Allied
Industies
(151)
Treatment Of Mixed Radioactive
Wastes
D. radiodurans
Radioactive Wastes
(152)
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humans consume a sufficient amount of contaminated seafood (phycotoxinsare odorless and tasteless),
intoxication occurs. Table (1) shows the recently used biosensor.
Table (1): Recently used biosensor
Examples of Field application of biosensor
Several genes encoding the OP-degrading enzyme have been identified and, with the benefit of molecular
biology technology, applied in whole cell biosensors as the recognition element (9) (100). For example,
genetically engineered p-nitrophenol (PNP)-degrader Pseudomonas putida JS444 and Moraxella sp.,
displaying organophosphorus hydrolase (OPH) activity, were constructed to selectively and sensitively
detect paraoxon, methyl parathion, parathion, fenitrothion, and ethyl p-nitrophenol thiobenzene
phosphonate (EPN) (101, 102, 103, 104, 105).
A microbial BOD sensor quipped with an oxygen electrode and activated sludge was developed and
successfully applied to the real-time monitoring of anaerobic reactors (106). Table (2) illustrates some
biosensor that used in field. Table (2): some biosensor that used in the field
Field application
Bacteria
Description
Reference
Determination of choline
Arthrobacter globiformis
Modifed oxygen electrodes
(122)
Determination of caffeine
Pseudomonas alcaligenes
Modifed oxygen electrodes
(123)
Analysis of genotoxicity
Salmonella typhimurium
TA1535 psk1002
With a screen
-
printed
(125)
NA (nalidixic acid) and (IQ)
E. coli
RFMuu3/PBR2TTs and
S.typhimurium ta1535psk1002
Micro chip
(126)
Detect Cd
2+
C.valgaris
Detection limit was a slow as
1ppbcd2+
(127)
Detection urea
Brevibacterium
ammoniagenes
In the ph sensitive polyanilineonapt
(128)
Detection of cephalosporin
group of antibiotics
P.aeroginosa
Phelectord modified
(129)
Determination Cu
2+
Circinella sp.
Modified carbon paste electrod
(131, 130)
Detection of
genotoxinmitomycin
E.
coli DH5a
A miniature flow
-
through optical
cell-based disposable fluorescent
microbial
(132)
Based on the respiration of cells, Pseudomonas sp. entrapped polycarbonate membrane modified oxygen
electrode was also applied to detect PNP which serves as the sole carbon and nitrogen source for the
bacteria (107). In addition, Moraxella and P. putida modified glassy carbon electrodes (GCE) were utilized
in the detection of E. coli in water based on the highly sensitive amperometric detection of PNP, a
metabolic product of E. coli, with the addition of p-nitrophenyl-ˇ-d-glucuronide (108).
The decrease of oxygen consumption rate due to the inhibition of bacterial respiration can also be utilized
to detect highly toxic chemicals. According to this mechanism, photosynthetic microalgae Chlorella
vulgaris modified screen-printed carbon electrode was reported for the continuous evaluation of the
toxicity of atrazine and DCMU (109).
Generally, the bacteria, which can uptake glucose, also possess the enzyme activity to metabolize other
carbohydrates, such as galactose (110) (111), catechol (112), mannose, and xylose (110). Selective
biosensors can still be developed for different sugars as long as the bacteria are adapted to the specific
analyze in advance through the selective cultivation. The qualitative and quantitative detection of
alcohols with high sensitivity, selectivity, and accuracy is required in many fields (113). Several
microorganisms which can metabolize ethanol with the consumption of oxygen, such as G. oxydans (114,
115), Pichia angusta (116), and Candida tropicalis (117) have been applied to the construction of ethanol
whole-cell biosensors. Moreover, a G. oxydans based amperometric biosensor with ferricyanide serving as
the mediator for the measurement of ethanol in FIA system was constructed (118). The experimental
results showed that the response was very stable during 72 h of continuous monitoring and the detection
limit was as low as 3.3M for ethanol. Another microbial biosensor based on G. oxydans for the selective
determination of 1,3-propanediol (1,3-d) in the presence of glycerol (Gly) in the flow system was also
reported (119). With the help of either ferricyanide or Osmium redox polymers, a good operational
stability was demonstrated by 140 h of continuous operation with a selectivity ratio (1,3-d/Gly) of 118 or
145, respectively.
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Microbial biosensors are prospective in the determination of some compounds, which is costly and time-
consuming by using traditionally analytical method. S. cerevisiae coupled with anamperometric oxygen
electrode was shown to be sensitive in the detection of thiamine (120) and L-lysine (121). Arthrobacter
globiformis (122) and Pseudomonas alcaligenes (123) modified oxygen electrodes were reported for the
determination of cholineand caffeine, respectively. A L-lactate selective biosensor was developed using
permeabilized, genetically engineered Hansenul apolymorpha immobilized on a graphite electrode with
phenazinemethosulphate as the free-diffusing redox mediator (124). Recently, a whole-cell biosensor on
the basis of recombinant Salmonella typhimurium TA1535 pSK1002 coupled with a screen-printed gold
electrode was designed for the amperometricanalysis of genotoxicity, and the result obtained therein was
consistentwith that obtained by standard SOS-umu-test (125). In another recent study, E. coli
RFM443/pBR2TTS and S. typhimurium TA1535 pSK1002 were integrated onto a microchip to fabricate
novel microfluidic whole-cell biosensors for the detection of two genotoxic compounds, nalidixic acid
(NA) and2-amino-3-methylimidazo4,5-f. quinoline (IQ), respectively (126). The detection limits of
10_g/mL for NA and0.31_M for IQ were achieved.
Demonstrate das OP compounds can inhibit the activity of acetylcholinesterase. A similar conductometric
biosensor using C. vulgaris as the sensing element was fabricated to detect Cd2+ and the detection limit
was as low as 1 ppb Cd2+ (127). Another conductometric biosensor was constructed by entrapping
lyophilized Brevibacterium ammoniagenes in the pH sensitive polyaniline on a Pt twin wire electrode to
detect urea (128). The catabolic activity of bacteria produced ammonia from urea, thereby causing an
increase of the local pH. The variation pH resulted in the change the resistivity change of the CP, which
was detected by the working electrode.
Recently, a potentiometric biosensor based on the pH electrode modified by permeabilized P. aeruginosa
was developed for selective and rapid detection of cephalosporin group of antibiotics (129). The
hydrolysis of cephalosporin, due to the enzyme activity of the microbial layer, was accompanied by the
production of protons near the pH electrode. The response came from the change of electric potential
difference between the working electrode and the reference electrode. Another potentiometric biosensor
for the identification of Beta-lactam residues in milk was also reported (26).
Recently, a voltammetric microbial biosensor for the determination of Cu2+was reported (130). The
biosensor was based on Circinella sp. modified carbon paste electrode. Cu2+ was pre concentrated on the
electrode and measured by CV, which was the interpreted as peak current to determine the concentration
of the target analyze. The detection limit of this biosensor could be a slow as 54nM Cu2+. Furthermore, a
modified stripping voltammetry was proposed by taking advantage of both bacterial adsorption on a
mercury surface and metal fixation capacity on A. ferrooxidans (131).
Martineau et al. reported a miniature flow-through optical cell-based disposable fluorescent microbial
biosensor for the detection of genotoxinmitomycin C (132).
Another bioluminescent microbial biosensor based on luxCDABE marked Acinetobacter sp. bacterium was
used to assay the toxicity of wastewater contaminated by heavy metals (133). In addition, a
bioluminescent biosensor with P. fluorescensHK44 pUTK21 recognition element was designed to reflect
the available fraction of naphthalene in soil since there was a linear relationship between the
luminescence from microbes and the naphthalene concentration (134).
CONCLUSION
All literature indicated that biosensor can be applied in the marine environment especially for detection
of pollutant such as: Crude oil, Heavy metal, insecticide and etc. Between all described biosensor the
biosensor that work based on bioluminescence better applied in the marine environments.
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CITATION OF THIS ARTICLE
Pariya Ahmadi B, Mehdi H.Microbial Biosensor for Marine Environments. Bull. Env. Pharmacol. Life Sci., Vol 3 [Spl
Issue V] 2014: 01-13
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and
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