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Anuradha Pandey (Dubey) and Madhuri Sharon, BAOJ Chem 2017, 3: 23: 029

BAOJ Chemistry

Review

BAOJ Chem, an open access journal Volume 3; Issue 2; 029

*Corresponding author: Madhuri Sharon, Walchand Centre for Research in Nanotechnology & Bionanotechnology, Walchand College of Arts & Science, WH Marg, Ashok Chowk, Solapur, 413006, Maharashtra, India, Tel: 91-8655723028; E-mail: [email protected]

Sub Date: July 18, 2017, Acc Date: August 07, 2017, Pub Date: August 08, 2017.

Citation: Anuradha Pandey (Dubey) and Madhuri Sharon (2017) Biolu-minescent Organisms. BAOJ Chem 3: 029.

Copyright: © 2017 Anuradha Pandey (Dubey) and Madhuri Sharon. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Bioluminescent Organisms

Anuradha Pandey (Dubey)1 and Madhuri Sharon1*

1Walchand Centre for Research in Nanotechnology & Bionanotechnology, Walchand College of Arts & Science, WH Marg, Ashok Chowk, Solapur, Maharashtra, India

Abstract

A general review of Bioluminescence is provided. It touches the reason for evolution of Bioluminescence and its different types; which is exhibited by various living beings. Bioluminescence is an inner glow that is due to cold light produced by that organism due to a chemical reaction occurring within a living being. In all known bioluminescent organisms the chemical reaction is based on a specific molecule called luciferin. This review encompasses study of bioluminescence exhibited by Firefly, Fungi, Bacteria, Fish, Crustaceans, Dinoflagellates, Deep-sea blooms and Plants. The Biochemistry and Molecular Genetics of Generating Bioluminescence by Photoproteins, Luciferase and Biochemical Mechanism of Luciferase has lead to the belief that in future there may be many environmental applications, feasibility of bio-lighting, bioluminescent immobilized systems and biosensors etc. Moreover, recent interest of Nanotechnologists in Bioluminescence has also been briefly touched on. Moreover, interest and dabbling of synthetic biologist with bioluminescence is also directing it to be a part of many emerging technologies and applications.

Keywords: Bioluminescence; Photoproteins; Luciferase; Firefly; Luciferins

Introduction

Bioluminescence is an inner glow exhibited by many organisms in nature. It has a surreal beauty that captures the imagination of scientists and the public alike. Bioluminescence spans all oceanic dimensions and has evolved from bacteria to fish, to powerfully influence behavioural and ecosystem dynamics. The natural bioluminescent world includes varied terrestrial creatures also such as beetles, fungi and bacteria. Bioluminescent organisms, with their inherent beauty and ease of detection, have attracted the attention of biochemists, physiologists, synthetic biologist and molecular genetics scientists. In this review we have touched upon efforts and attempts of scientists from diverse field in understanding and utilizing this novel property of Bioluminescence for various applications.

What is Bioluminescence?

Bioluminescence is light produced by a chemical reaction occurring

within a living organism. It is a type of chemiluminescence, which produces ‘’cold light’’ Cold light means it generates less than 20% thermal radiation, or heat.

The Bioluminescent chemical reaction in all known organisms is based on a specific molecule called luciferin. It is a molecule that reacts with oxygen, produces light. There are different types of luciferin, which vary depending on the animal hosting the reaction. Many organisms also produce an enzyme called luciferase, which catalyzes or speed up the reaction. This reaction in Bioluminescent animals are controlled when they light up by regulating their chemistry and brain processes depending on their immediate needs, whether a meal or a mate. Some organisms even bundle the luciferin with oxygen in what is called a “photoprotein”-like a pre-packaged bioluminescence bomb-that is ready to light up the moment a certain ion (typically calcium) becomes present. They can even choose the intensity and colour of the lights. The detailed chemistry of Bioluminescence is mentioned later in this review.

Why Bioluminescence Evolved?

Bioluminescent organisms are widely distributed, mostly inhabiting terrestrial and marine ecosystems. These organisms develop the capacity of bioluminescence as per their habitat and survival needs. For example, many luminescent bacteria that exist planktonically, as gut symbionts, as saprophytes or parasites or in specialized light organs of certain fishes and animals in marine environments.

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These bacteria whether they are free living or in association with higher organisms, utilize bioluminescence for vital functions ranging from defence to reproduction.

So far as terrestrial or higher bioluminescent organisms are concerned they use this property mainly for attracting prey, intraspecies communication and escape from predators.

Bioluminescence is rarely found in fresh water. Most scientists point to two reasons why bioluminescence is not evolved in freshwater; one is that many freshwater habitats have not been around as long as marine habitats neither they have biodiversity of oceans and to top it all evolution is rather slow. Second, freshwater species wouldn’t really benefit from bioluminescence. Freshwater habitats are often murkier, and deepwater species use other adaptations (such as a catfish’s sensitive “whiskers”) to hunt and defend in the environment.

Bioluminescence is generally produced by the oxidation of a light emitting molecule Luciferin in the presence of a catalyzing enzyme- luciferase or photoprotein. There are some non symbiotic luminous organisms which posses the gene for their luciferase or photoprotein. Symbiotic luminescent organisms that have evolved uses these microbes to develop specialized light organs. Bioluminescent molluscs have evolved many times to make light in different ways as per the study done by researchers. Bioluminescence has evolved many times, however it is difficult to calculate the number of times it has evolved independently. Some of the bioluminescent functions have not yet been explored. To generate a rough estimate, many studies summed-up few distinct light-producing chemical mechanisms across the monophyletic lineages, to estimate that bioluminescence has evolved a minimum of 40 to 50 times, among extinct organisms. Since, the ability to make light has evolved many times, it suggests that it is important to organisms, and also that its evolution must be relatively easy. While counterintuitive, this may be partly attributed to readily available light-emitting luciferin in both luminous and as a way to trigger light emission [1,2]. Bioluminescence can more readily evolve if naturally occurring antioxidant molecules are present in an organism, and if light is produced as a by-product of those molecules due to chemical activity in the scavenging of reactive oxygen species, as hypothesized by a study [3,4]. Studies show that dietary linkages also suggest that to some extent luminescence is a post-Cambrian development, since it had to arise in the predators after the synthesis of luciferins evolved in the prey. The fossil record and dates of phylogenetic separation estimated by molecular clocks may help to bracket the dates of luciferins utilized within particular groups, but it is presently difficult to narrow the range within 100 million years. Two, even lineages of ostracod crustaceans (Halocyprida and Myodocopida) that use two different luciferins are thought to have diverged more than 400 million years ago [5],

suggesting this as a maximum age for at least one of the known luminescence systems. The fish order Stomiiformes is found to be bioluminescent throughout and is thought to have originated in the Albian age of the early Cretaceous, about 100 million years ago [6]. The stomiids are certainly well established by the occurrence of a convincing hatchet fish from 12 million years ago, which appears remarkably like its modern counterparts [7].

The multifarious evolution of bioluminescence is also underscored by its widespread distribution throughout the ocean, from the surface to the deep sea and from the poles to the tropics. For most of the marine animals their primary visual stimulus comes from biologically generated light and not from sunlight.

Different Forms of Bioluminescence

As observers, we typically encounter different types of bioluminescence in organisms. It is rather difficult task to categorize them under one sub-heading because the differences could be caused by or due to a response to environment by a physical disturbance that may induce just a flash or it may be continuous or blinking luminescence. There are differences in spectral properties of luminescence thus exhibiting different colours. Different organisms have different light emitting tissues. Some bioluminescent differences are observed due to the physiological mechanisms that controls the bioluminescence

In a natural context, however, the emission of light is closely controlled by chemical and neurological mechanisms. Animals can turn their photophores on and off, but they can also modulate the intensity, colour, and even angular distribution of light. These control mechanisms often involve calcium ions and other standard neurotransmitters. In dinoflagellates, control over light emission begins with a physical stress that deforms the membrane [8] and involves a cascading series of triggers, including G-protein-coupled receptors [9], calcium ions [10] and ultimately H+, which triggers the release of luciferin from its binding protein, as revealed in many studies. This sequence of events occurs with less than 12 ms latency, as measured with great precision on single cells using a micro-fluidics setup [11].

It has been found that bioluminescence in many organisms is under nervous control, which can provide a visual map of the conduction velocities of the nervous system. This has been proved by Panceri [12] with his experiments on scyphozoans and it was continued with application to the nerve net of the sea pansy Renilla and to the epithelial conduction of hydrozoans [13]. This is a natural analogue to the ways that calcium-activated photoproteins currently are used in research to create bio-optical sensors and to illuminate cellular processes [14,15].

Dinoflagellate’s luminescence and the light from other organisms can be tied to a circadian rhythm, in which the luciferins are



oxidized and rebuilt with each day/night cycle [16]. Other bioluminescent organisms living in illuminated environments may also require a period of dark acclimatization before they will begin to luminescence; in the case of ctenophores, this is due to direct photo-degradation of their photoproteins [17]. In most deep-sea animals, however, the ability to produce light is present.

Scientists reveal, Bioluminescence is an extremely effective way for invertebrates to communicate to organisms much larger and potentially far away and this may help explain its prevalence. Depending on the conditions, a bioluminescent flash can be seen from tens to hundreds of meters away [17,18]. Even a 0.5 mm long single-celled dinoflagellate can send a signal to a large fish 5 m away i.e. equivalent to a 2 m tall human being able to communicate over a distance of 20 km. Chemical cues, while certainly playing important and overlooked roles in the sea [19,20], perform a different set of functions than optical or acoustical signals; they do not diffuse rapidly enough to send the acute signals across distances that are possible through bioluminescence. Chemicals do have the advantage, however, of operating between non-visual organisms, where bioluminescence is ineffective. Acoustical signals are transmitted extremely effectively in the sea [24], but they have the drawback of being relatively non-directional, and many planktons lack the hard or firm body parts or gas bladders required to generate sounds [21-23] Thus bioluminescence is one of the most effective ways for a small organism to communicate efficiently to a much larger organism in the sea.

Some other light emitters like bivalve Pholas [24], polychaetes, including the polynoiid scale worms [25], chaetopterid tube worms and syllid fireworms have also been recorded and studied [26]. A notable addition to the list of marine luciferins came with the elucidation of the chemistry of the hemichordate Ptychodera flava [27]. The hypothesized light emitter, which operates with the involvement of peroxide and riboflavin, has a unique simple symmetrical structure. There is a need to study different types of luciferins, especially found in vermiform phyla, echinoderms and mollusc.

Despite the taxonomic diversity of bioluminescent organisms in the marine environment, their spectral properties are mainly constrained to blue green wavelength, i.e. around 470 nm, with few exceptions. In certain organisms the emission of luminescent light is modified by the presence of fluorescent proteins or fluorescent compounds [28-31]. These spectral properties are closely linked to the visual systems of marine animals, both driving the adaptation of colour sensitivity [32-35] and being driven by the qualities of the marine environment [36-39]. In planktonic cnidarians and ctenophores, bioluminescence spectra was found to be shifted to shorter wavelengths as the species depth of occurrence increases [40], which corresponds to the trend seen in fish visual pigments

[41]. Most of the species found at the shallow depths use green fluorescent protein as part of their luminescence system, and this modification is mostly observed among deeper-living species [42].

It has been found that most of the deep sea fish (Figure 1c) have monochromatic colour sensitivity ranging near the blue green wavelength [43], with few exceptions that emit red light themselves [44], can detect longer wavelengths than just blue or green light [45-47]. Squids can also have chromatic vision through the presence of multiple visual pigments [48,49], and some, in particular Vampyroteuthis infernalis, have lenses capable of acute vision [50], which would lend themselves to discriminating bioluminescent sources against a dim or dark background. This review thus focuses on various forms and sources of bioluminescence and its exploitation for future applications.

Firefly Bioluminescence

The first living organism that comes to our mind is glowing fireflies (Figure1a and b) in rainy season. Firefly is an insect belonging to family Lampyridae with about 2000 species. Their habitat is wet decaying wood, marshes, river side etc. of tropical and temperate areas.They produce yellow, green or light red luminescence from their lower abdomen to attract mates and prey.

Of the known bioluminescent systems, firefly luciferase is particularly well studied and characterized due to their common use as reporter genes. The luciferase from Photinus pyralis was the first to be chosen for creating glowing transgenic plant.

The protein was expressed in tobacco plants, from a cauliflower mosaic virus promoter, and the resultant genetically modified organisms (GMOs) were then watered with luciferin. Light emission was seen when plants were placed on photographic film with the brightest light tracing out the water transporting vessels of the leaves and stem [51]. The reaction is known for its high quantum yield, emitting a strong yellow/green light [52]. This might make the system a good candidate for bio-lighting applications; however, despite the luciferase gene being sequenced in 1987, the genes for production of its substrate remain unknown. With sequencing ever cheaper and the abilities of researchers to annotate genomes and characterise parts improving, this will change but for now the reaction in transgenic organisms requires addition of expensive luciferin substrate.

The presence of an enzyme for recycling the luciferin substrate was suggested in 1974 by Okada et al [53]. The researchers discovered that radioactively labelled oxyluciferin and 2-cyano-6-hydroxybenzothiazole (CHBT) gets converted into luciferin when injected into living fireflies. Thirty years later the protein involved was identified and named luciferin regenerating enzyme (LRE) [54]. This enzyme was shown to recover D-luciferin with



the addition of D-cysteine. Oxyluciferin can block the firefly luciferase active site so is a competitive inhibitor of luminescence. Its removal by LRE improves light output in vivo with the addition of D-cysteine prolonging output even further [55]. This is a step towards sustained light output in a modified organism from the firefly system. Genes for de novo luciferin biosynthesis might only have to produce a little of this substrate and it could then be recycled near indefinitely via the LRE cycle. Most organisms do not produce D-cysteine however: it is acutely toxic to mammals and a strong inhibitor of bacterial growth so could pose difficulties for establishing this LRE cycle in alternative organisms. Possible recycling via L-cysteine has also been reported [56] and though not yet shown in synthetic organisms this may in future be the pathway of choice for recycling luciferin in bio-lighting. A different reason for employing the firefly system in bio-lighting is the broad spectrum of colours possible. It was noted that firefly luciferase in vitro were pH sensitive and their colour could vary from yellow/green to red depending on the conditions. In vivo to a range of colours are possible: mutants have been produced, which show different spectral outputs at the same physiological pH [57]. Single amino acid changes were introduced in the Japanese firefly (Luciola cruciata) luciferase, another popular reporter protein. These substitutions change the peak wavelength of light output visibly altering the colour of luminescence.

Fungal Bioluminescence

Like firefly bioluminescent fungi are also found in both tropical and temperate zone. Fungal bioluminescence, colloquially known as `foxfire’ is an impressive sight in wet woodland. More than 75 terrestrial bioluminescent fungal species have been described so far. All of them belong to order Agaricales, the order of Mushrooms (Figure 2c).

The biochemistry is relatively poorly understood. Possible luciferins have been discovered yet the pathways for their production are still unknown. No luciferase enzymes have been sequenced, as the existence of a luciferase was only recently confirmed [58]. Previous theories suggested light was produced by an un-catalysed reaction between metabolites. This lack of information is a barrier for consideration in synthetic biology applications. There may be wonderful luminescent genes and systems hidden in fungi, but sadly genetic and biochemical understanding must catch up to that of insects and bacteria before they can be utilised. There are similar knowledge barriers for the systems from the numerous other luminescent organisms. Considered species include dinoflagellates, responsible for luminescent waves sometimes seen in tropical waters, Cnidaria (the phylum containing jelly-fish and anemones) such as the sea pansy Renilla reniformis, and copeptide crustaceans (Figure 1f) such as Gaussia princeps. For these organisms the

Figure 1: Few Luminescent organisms (a) Firefly during day and (b) Firefly glowing in night while flying(c) A deep sea fish and (d) A deep sea firefly Squid, both showing Photophores (e) Comb jelly and (f) Some Bioluminescent Crustaceans (Source :- ati.com)



luciferase enzymes and their substrates-a chlorophyll derivative in dinoagellates and coelenterazine in cnidarians and copepods are well known but the rest of their biochemistry is not.

The lack of described biochemical pathways for substrate production and knowledge of the genes involved prevents experimentation in synthetic biology applications for now. But this will change as our understanding of the natural world grows. Our curiosity to learn about living systems in nature not only drives us to new discoveries but it also equips us with the knowledge to design and build new living systems.

Bacterial Bioluminescence

Aside from fireflies, luminous bacteria have also received considerable attention. All luminescent bacterial species are Gram-negative, non-spore forming and motile. They are the abundant and very widely distributed light-emitting organisms. They can be found as free living species in the ocean, as saprotrophs on dead marine organisms, as symbionts in the light organs of fish and squid (Figure 1d) and many other ecological niches.

Among prokaryotes, light production is known only from the Eubacteria, specifically Gram-negative γ-proteobacteria, and not from Archaea. The best-studied symbiotic bacteria are in the genus Vibrio including the predominantly free-living species V. harveyi (sometimes called Beneckea harveyi), although the genus Shewanella also includes a bioluminescent species [59]. A study recently showed that many new strains of luminous bacteria, that has traditionally been called Photobacterium phosphoreum, are present in the deep sea [60], and many of them actually represent diverse assemblages [61]. Although there are many exceptions, Vibrio fischeri, often called Photobacterium (Figure 2a and b) is part of the species-complex typically involved in symbiosis with sepiolid and loliginid squid and monocentrid fishes, while Photobacterium leiognathi and relatives are primarily symbionts for leiognathid,

apogonid, and morid fishes [62]. Known luminescent bacteria, are found in the genera Aliivibrio, Photobacterium, Alteromonas, and Photorhabdus.

The lux operon, the bacterial enzymatic light emission system is highly conserved amongst the various species of bioluminescent organisms and is commonly represented by lux CD-ABEG. The light emission reaction mainly utilizes flavin mononucleotide and a long chain aldehyde, a derivative of fatty lipid biosynthesis as substrates. The genes Lux A and Lux B encode α and β subunits of bacterial luciferase, lux C, D, E encode enzymes for synthesis of aldehyde substrate, lux G codes for flavin reductase that participates in flavin mononucleotide turnover [63]. This inbuilt light emission system is being exploited by researchers in synthetic biology. The operon contains all the genes necessary for bioluminescence, production and cycling of the substrate plus the light producing reaction all together. This makes its study and transfer to certain new hosts considerably easier. It may also be a limitation however, eukaryotic nuclear Deoxyribo Nucleic Acid (DNA) does not contain operons and the cytosolic ribosomes cannot usually process them so the genes require considerable refactoring for these hosts. Bioluminescent Escherichia coli were produced in the 1980s with the operons from Vibrio harveyi and aliivibrio fischeri [64]. Early experiments with Agrobacterium mediated transformation involved only the luciferase enzyme. Vibrio harveyi lux A and B were expressed in transgenic tobacco and carrot cells and shown to correctly assemble into functional luciferase with light produced from extracts with substrates added [65].

Transgenic auto luminescence in a higher eukaryote was only achieved decades later with the refinement of ballistic transformation methods. The entire Photobacterium leiognathi lux operon was transformed into the chloroplasts of tobacco plants and produced luminescence [63]. In the same year the whole operon-with genes chosen from Photorhabdus luminescens and V.harveyi

Figure - 2: Cultures of bioluminescent bacteria Vibrio fischeri growing on nutrient slants in (a) test tubes and (b) on petridish; Bioluminescent fungus Panellus stipticus growing on a log (b) A glowing plankton Dinoflagellate (Source : ati.com)



-was expressed in mammalian cells but required considerable refactoring, codon usage was re-optimised and the six genes were split into three bicistronic pairs with a viral internal ribosome entry site (IRES) between the pair. This allowed weakly autoluminescent HEK293 cells (a human cell line) to be produced. Other attempts at bio-lighting have utilised the V. fischeri lux operon. When expressed from an arabinose inducible promoter in E. coli, and activated by the addition of L-arabinose, in large flasks produced just enough light to read [55].

Deep-Sea Bioluminescence Blooms

The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Many observations provide a consistent and rapid connection between deep sea convection and bathypelagic biological activity, expressed by bioluminescence. Tamburini et al [66] have shown concern over impact of environmental changes such as global warming scenarios enhancing ocean stratification thus affecting the monitoring and understanding of deep-sea ecosystem shifts.

The deep-sea ecosystem is unique because of its permanent darkness, coldness, high pressure and scarcity of carbon and energy to sustain life. Most of its biological activity relies on the arrival of carbon in the form of organic matter from surface waters. Ninety percent of the numerous pelagic organisms that inhabit the deep ocean are capable of emitting light through the chemical process of bioluminescence, which appears to be the most common form of communication in this remote realm. Deep-sea bioluminescence is also viewed as an expression of abundance and adaptation of organisms to their environment. Marine bioluminescent organisms include a variety of distinct taxa [66]. When stimulated mechanically or electrically, eukaryotic bioluminescent organisms emit erratic luminous flashes, and also spontaneous flashes to attract prey and mates for recognition of congeners or for defence purposes. In contrast, luminescent bacteria are unaffected by mechanical stimulation and can glow continuously for many days under specific growth conditions. Bioluminescent bacteria are observed in marine waters as free-living forms, as symbionts in luminous organs of fishes and crustaceans and attached to marine snow aggregates sinking through the water column. A strong bioluminescence is observed by colonies of bacteria during micro algae blooms.

Other Bioluminescent organisms

Bioluminescence is used by some animals to aid hunting, mating, to send a warning (sometimes of toxicity) to predators, to startle, for counter illumination and also attacking a prey. Some of the common animals that show bioluminescence are: Jelly fish that

creates its own light in deep underwater, Squid that harbours bioluminescent bacteria, Angler fish which uses bioluminescence to attract fish and tiny Crustacean (sea shrimp) which produces blue light are already discussed. Some other animals are discussed below.

Dinoflagellates

Dinoflagellates are unicellular organisms with 2 flagella (Figure 2d). Next to fireflies, dinoflagellates are the most commonly encountered bioluminescent organism. They typically cause the sparkling lights in the water seen by sailors, swimmers, and beachgoers, and they produce the “bioluminescent bays” which are tourist destinations in Puerto Rico and Jamaica. These protists can be autotrophic (photosynthetic) orheterotrophic, feeding on other phytoplankton and prey. In large numbers, some species may form potentially toxic red tides, typically during a stratified calm period after an influx of nutrients. Studies reveal that there are at least 18 luminous genera [67] of Dinoflagellates including Gonyaula Noctiluca, Protoperidinium and Pyrocystis, that exhibit their ability to luminesce, and allocate energy to bioluminescence before growth, although luminescence serves secondary to the ability to swim [68].

Fish

Bioluminescence is observed in at least 42 families across 11 orders of bony, including one family of sharks. In contrast with invertebrate taxa, most of these groups use bacterial symbionts for light production, including the anglerfishes, flashlight fish like Photoblepharon spp, and shallow ponyfishes like Leiognathus spp. The other luminous fishes have intrinsic luminescence using either coelenterazine [69], ostracod luciferin, or some other uncharacterized light-emitter. Fish photophores are often highly modified and adapted to control not only the intensity of light but its angular distribution, according to their particular function [70].

Myctophids, or lantern fishes are extremely abundant in the mid-water, migrating near the surface at night. They have small photophores pointed downward and to the side, as well as large photophores on the tail, which can produce bright, fast (1×1011 photons s-1 for <400 ms) flashes. The genus Diaphus, which feeds actively on copepods and amphipods [71], has prominent forward-facing photophores, which may be used to illuminate or induce fluorescence in their prey. If they use these headlights for hunting, it may also make them susceptible to visual attraction, as this genus was the most abundant in the diet of stomiid dragonfish [72].

The order Stomiiformes I.E. hatchet-fishes, dragon-fishes includes some of the most elaborate arrangements of photophores, including barbells, ventral arrays, and red and blue suborbital photophores [31]. Within the family Stomiidae, there are several new species,



which are distinguished partly on the basis of their barbel and light-organ morphology [73-76]. Their diversification could be tied to feeding ecology, since stomiid species have a fairly high degree of prey specificity, and the favored prey of various species span copepods, euphausiids, decapod shrimps, fishes, and squid [77]. Among the most interesting members of this group are the Malacosteinae, which are well known for having the unique ability to produce and detect long-wavelength red light [78,45,31]. What is curious about this group is that Aristostomias and Pachystomiaseat fish, as expected, but Malacosteus, despite its large teeth, few gill-rakers, and no floor to its mouth, feeds frequently on copepods with only occasional meals of large fish [77]. Although the three genera share unique visual and bioluminescence abilities, Malacosteus also differs in achieving its long-wavelength sensitivity by a distinct mechanism requiring chlorophyll as a sensitizer [43]. Since vertebrates cannot synthesize chlorophyll, Malacosteus gains its visual abilities through its diet of copepods, which contain phytoplankton-derived pigments in their guts. The ability to produce long-wavelength light may turn out to be more widespread than originally thought, since other stomiids have red suborbital photophores that appear morphologically similar to those of the Malacosteinae.

In the order Chondrichthyes, the Squalidae are a family of luminous lantern sharks that use ventral counter shading for both defensive and offensive purposes. In the evolutionary jockeying for selective advantage that occurs between animals that emit and those that detect bioluminescent light, fish and squid have developed additional ways to improve their perception.

Crustaceans

Crustaceans include familiar animals such as crabs, lobsters, cray-fish, shrimp, krill etc. Mostly crustaceans are free-living aquatic animals, but some are terrestrial and parasitic also. However there are many planktonic crustaceans also which are bioluminescent and they use species-specific luciferases with at least three different types of luciferins. It has been found that crustaceans rival molluscs and polychaetes, Euphausiids, or krill use the same luciferin as di-noflagellates [79,80], strongly suggesting a dietary connection. The lower surface of their body, have light organs which they use for counter illumination, and some species also have two small light organs on their eyestalks. These serve as feedback mechanisms for determining how well their ventral photophores are matching background light. Like most photophores, these are under nervous control that involves serotonin moderated by nitric oxide [81].

Plant Bioluminescence

Plants do not exhibit strong bioluminescence. However, way back in 1935 Lepeschkin [82] demonstrated ultra week bioluminescence (UWL) in plant tissues using photographic technique. UWL has

extremely low quantum efficiency in the range of 10-14 to 10-9 photon per activated molecule [83]. It was observed that there is a relation between morphogenesis and UWL emission [84]. In germinating seedlings root growth rates were directly correlated with UWL intensity. Meristematic zone of roots showed highest UWL, whereas elongation zone of the roots was considerably weaker. Although glowing bioluminescent plants don’t exist naturally, but efforts have been on to produce genetically engineered plants using bioluminescent fungi or firefly. The first bioluminescent plant was made in 1986, with the addition of luciferin from firefly. Now a glowing a bioluminescent plant is available of the shelf in the market. Some of the successful efforts are discussed below:

Generation of Autoluminescent Nicotiana Tabacum Plants

Krichevsky et al [85] in their research work on generation of auto-luminescent Nicotiana plants have generated two independent lines (Figure 3A and B) of Nicotiana tabacum trans-plastomic plants, carrying the bacterial lux operon from Photobacterium leiognathi i.e.

where the lux operon was integrated into the rps12/TrnV locus of the chloroplast genome

where the lux operon was integrated into a more transcriptionally active TrnI/TrnA locus [85]

These plants were produced by cloning lux operon, containing the lux CDABEG genes under the control of the tobacco plastidal Prrn promoter into the plastid transformation vector pCAS3, which carries a spectinomycin resistant selection marker aadA, resulting in pCAS3-aadA-LUX vector. Homologous recombination sites for integration into rps12/TrnV or TrnI/TrnA tobacco plastid genome loci were inserted to flank the aadA-lux expression cassette, resulting in pCA3-LUX-rps12/TrnV or pCA3-LUX-TrnI/TrnA vectors, respectively, and trans-plastomic plants were generated using standard micro bombardment methods and selection on spectinomycin- supplemented media [86,87].

To differentiate or rather identify the true transplastomic plants from small ribosomal RNA (rrn16) spontaneous mutants, which are also spectinomycin resistant [85,87] newly initiated shoots were analyzed using junction PCR. The 2.35-kb fragment amplified using primers 78 and 104 and the 2.45-kb fragment amplified using primers 79 and 46 are diagnostic of integration of the entire expression cassette integration into the rps12/TrnV locus (Figure 3B). Further they amplified luxB and luxC to confirm the presence of the lux operon within the transplastomic genome (Figure 3B). The relatively small (155 kb) plastid genome in tobacco is present in thousands of copies per cell [89]. Integration of aadA and the lux operon genes in the LUX-rps12/TrnV genome, and the absence of non-transformed wild-type ptDNA copies has been confirmed by DNA gel blot analyses using plastid targeting sequences, aadA and



lux gene probes.

Figure-3: Autoluminescent tobacco seedlings. (A) illustrates the PCR products predicted to arise from transplastomic plants generated using pCA3-LUX-rps12/TrnV and (B) demonstrates the results of this junction PCR analysis (Krichevsky et al., 2010)

Similarly trans-plastomic plants which were transformed with the pCAS3-LUX-TrnI/TrnA plasmid have been identified using junction PCR primers specific for TrnI and TrnA, luxB and luxC. Integration of aadA and the lux operon genes in the LUX-TrnI/TrnA genome, and the absence of non-transformed wild-type ptDNA copies has been confirmed as described for the LUX-rps12/TrnV plants. The trans-plastomic plant lines do not exhibit detectable phenotypic changes in their morphogenesis.

First Autoluminescent glowing plant was first produced by Krichevsky et al [85] using lux operon-encoded enzymatic pathway for light emission. This approach can be applied to any plant species and can be further modified using different promoters so as to enhance the luciferase substrate levels in the cell, or enhance the luciferase catalytic activity [89]. The colour of the emitted light can also be modified via luciferase mutagenesis [90], and glow in specific plant organs can be achieved through the use of chloroplast-targeted nuclear re-encoded transcription factors expressed from tissue-specific promoters. Thus, their findings not only enhance our understanding of the fundamental biological processes and expression of complex and fully functional multi-enzymatic

pathways in plant plastids, but also are useful for floriculture industry, particularly since plastid DNA is maternally inherited in most flowering plant species [91], substantially reducing the risks of transgene escape into the environment.

Biochemistry and Molecular Genetics of Generating Biolumi-nescence

In the laboratory, bioluminescence has been central to techniques in biochemistry and molecular genetics for many years. Bioluminescent light is generated as a result of energy released during a chemical reaction. In most cases, this reaction is the oxidation of a light-emitting molecule, a luciferin. The reaction rate for the luciferin is controlled by an enzyme luciferase or another photoprotein, a luciferase variant in which factors required for light emission (including the luciferin and oxygen) are bound together as one unit.

Photoproteins

Photoproteins bind an ion or cofactor, such as Ca2+ or Mg2+ which causes a conformational change in the protein to produce light. This provides the organism a way to precisely control light emission.

Bacterial luminescence involves a two subunit luciferase and the oxidation of reduced flavin mononucleotide (FMNH2) along with a long-chain aldehyde. The genetic lux cassettes that are responsible for light production [92] and the conditions required for luminescence are now well understood [93], leading to numerous biotechnological applications [94,95].

Bacteria communicate with one another by producing chemical that serve as signal molecules. In higher organisms, the information supplied by these molecules is essential for synchronizing the activities of large groups of cells. In bacteria, the chemical communication involves to produce, to release, to detect, and responding to small hormone-like molecules termed auto inducers, This process which is termed quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behaviour is utilized in bioluminescence: milky seas consisting of widespread luminescence [96,97].

Luciferase

Lux operon encodes genes for self regulation and production of luminescent proteins such as luciferase (Figure 4). Luciferase is a heterodimer of 77 KDa, coded by two similar genes is activated by FMNH2 reacts with O2 producing a compound that will form a highly stable complex with the aldehyde that decays slowly by emitting luminous energy due to the oxidation of the substrates [98].

The aldehyde required for the reaction is synthesized by an enzymatic complex coded by three different genes present in all bioluminescent bacteria, luxCDE. This substrate is derived from



the fatty acid pathway and can be recycled after each reaction. The three proteins that form the enzymatic complex are a reductase, a synthetase and a transferase that were identified through radioactive labelling. Other proteins involved in the reaction are only present in certain species of bacteria. For example, lumazine and yellow fluorescent protein (yfp) are two different proteins present in Photobacterium phosphoreum and in some strains Vibrio fisheri respectively. These proteins are accessory proteins because they are not crucial for the reaction to take place. The protein (lumazine) shifts the colour of light to shorter wavelengths, thus increasing the energy of the emission. The second protein shifts the light produced to such a degree that the colour changes to yellow (540nm) [99].

Figure -4: Structure of firefly luciferase

The characterization of the lux operon was carried out by the cloning of a 9 kb DNA fragment obtained from V.fisheri that was transformed into E. coli. Successful expression was seen when E. coli was able to emit light in the manner as V. fisheri. The studies of Mutational analysis showed five crucial genes required for the reaction (luxAB and luxCDE) (Figure 5).

Figure - 5: Five different point mutant firefly luciferases and the wild type (second from right) expressed in E. coli with luciferin added to the media (Courtesy: Sanderson et al., 2010).

The genetic organization was determined i.e. an operon, a diverging with a left and right side containing two different promoters that transcribe in opposite directions [100,101]. The lux I gene at the start of the right side of the operon produces a small peptide that works as an auto-inductor that triggers the synthesis of the structural genes, luxCDABE, in that same order (Figure 6).

Figure– 6: Bioluminescence in bacteria is produced by the expression of lux genes. The Vibrio fischeri lux operon is organized in two different arrangements: from luxI to luxG are expressed downstream and opposite to luxR .

All genes in the right side are closely linked to each other, less than 50bp separates them. Next to luxE there is an additional gene that is present only in marine species not crucial for the reaction but its function is still unknown. To the left side of the operon a gene is present that codes for another regulatory protein, a repressor with dual function in all bioluminescent strains. This repressor has one site that interacts with the DNA and a second site that binds to the small peptide (luxI). When autoinducer is attached, it forms a complex that promotes transcriptional activation in the right region by positive regulation. When the autoinducer concentration is not high enough, the repressor is continuously produced. Thus, inhibiting the transcription of structural genes by binding to operator is located in the control region. This 218bp intergenic region between the left and right border of the operon works as the



control region. It contains the DNA binding site of the repressor and a site for camp receptor protein (CRP) [102].

Biochemical Mechanism of Luciferase

The mechanism of firefly luciferase provides insights into several interesting features of the luciferase-catalyzed reaction. Like the homologous acyl-CoA synthetases, luciferase can catalyze thioester formation between CoA and luciferin, using LH2-AMP as an intermediate. In the cross-linked luciferase, the pantetheine tunnel seen in other adenylating enzymes is intact and leads to the C4 carbon of adenylating enzyme. Luciferase contains in the A8 motif a conserved glycine residue Gly446 that lines this tunnel. In the thioester-forming enzymes of this family, this glycine forms a distorted β-sheet interaction with the amide nitrogens of the pantethene moiety and likely facilitates CoA binding in this alternate reaction. A G446I mutation in luciferase specifically impairs the oxidative reaction suggesting that O2 approaches the intermediate through this same tunnel. Side chain rotation of His245, further expands the tunnel for access to C4.

Figure 7: Schematic representation of Luciferase reporter mechanism

The structure also provides an explanation for the stereochemical requirements of luciferase. It is reported that while the enzyme is able to catalyze thioester-formation between CoA and both d- and l-luciferin, only d-luciferin is able to support the generation of light. This observation is explained by the accessibility of the carbonyl of the adenylate of both diastereomers to the pantetheine tunnel to be attacked by the CoA thiol. In contrast, the C4 proton must be available for removal, possibly as a hydrogen atom (Figure 8), in order to initiate the oxidative reaction required for the

generation of light. The structure demonstrates that the adenylate with l-luciferin would project the C4 hydrogen toward the core of the protein and away from the pantetheine tunnel, preventing proton or hydrogen atom abstraction [103].

Figure 8: Mechanism of Firefly Bioluminescence

Various Applications of Light Emitting organisms

In the intervening millennia technological advances have brought a succession of new approaches to lighting, from the grease lamps of antiquity, to candles, to the invention of the kerosene lamp in the Arab world. The nineteenth century saw the use of gas, then electricity, to emit brighter and cleaner light, and the twentieth has seen the refinement of electric lighting to be more efficient through the use of fluorescent bulbs and light emitting diodes. Alongside this growth in our technology for producing light has come a range of new applications. The most obvious reason for artificial light is to illuminate dark spaces so that we may see (e.g. street-lights). But our uses go well beyond this-light has long been important for aesthetic purposes in culture and art (crowds line up to see



fireworks fly into the sky). We also use it to convey information to humans (traffic lights, television screens), and now even to convey information between machines (optic fibres). This review examines the contributions that synthetic biology may make to each of these areas in turn.

Light is a crucial component of many systems in conventional engineering, and there is no reason to suspect that this will change as we begin to engineer biological systems to tackle the challenges humans face. Thus we can expect bioluminescence to have a key role to play in the continuing development of synthetic biology.

Various physicists, biologists have been focussing their attention to utilize bioluminescence in the near future for various applications-few of which are illustrated here.

Applications in Detecting organic Pollutants

Bioluminescent organisms are being researched and exploited for many applications. Luciferase systems and bacterial bioluminescent genes (lux genes) are widely used in the field of genetic engineering in environmental biotechnology as genetic reporters and contaminant biosensors, respectively [104]. Luciferase systems have also been exploited for biomedical research purpose using bioluminescence imaging. The symbiotic relationship of Vibrio with numerous marine invertebrates and fish, especially the Hawaiian Bobtail Squid (Euprymna scolopes), are key experimental models for symbiosis, quorum sensing, and bioluminescence.

When real samples are tested by Bioluminescent Bacteria (BLB), the responses obtained by these assays are usually compared with those supplied by different biotests, and vice versa. As an example, the sensitivity of an Early Life Stage (ELS) test studying survival, growth and histopathological changes in seabream (Sparus aurata) yolk sac larvae in determining simazine, a s-triazine herbicide, was compared with that of the commercial kit “Microtox” [105]. Simazine did not exert any significant toxicity to the marine bacterium V. fischeri at the tested concentrations, while the survival of the larvae was significantly reduced. On the other hand, luminescent data are often confirmed by instrumental analysis. BLB assays, using various strains of Vibrio sp., were developed as microplate format to evaluate the impact of the residues of veterinary pharmacological treatments on environmental and human health [106]. The bacterial responses, to pure antibiotic solutions and to residues extracted from excreta of treated animals, were compared with the direct quantification by High-performance liquid chromatography (HPLC) analysis, obtaining good correlation values. Quinolones are one of the most important groups of synthetic antibiotics used in aquaculture. The toxicity of a single of such molecules and of a mixture of ten quinolones was studied using a long-term inhibition assay on V. fischeri, an organism very sensitive to these substances. The EC50 values ranged from14µgL−1 for ofloxacin to 1020µgL−1 for

pipemidic acid [107]. The photoinduced toxicity of various kinds of compounds, by photosensitization or by photomodification, is a phenomenon that requires to be directly evaluated in the environment. A method for measuring photoinduced short and long-term toxicity of polycyclic aromatic hydrocarbons (PAHs), based on inhibition of luminescence and growth of V. Fischeri is available as per several research studies [108,109]. The short-term assay photosensitization activity is immediate and it detects toxicity of chemicals that are taken up rapidly whereas the long-term assay identifies chemicals whose rate of assimilation is slow and it requires time for photoinduced effects to be realized. For several PAHs, the short-term assay failed to reveal photo-induced toxicity that is evident only in the long-term assay. Eco-toxicity tests [110] on photolytic degradation derivatives of chlorinated paraffin showed that by the green algae test no inhibition of cell growth was observed. On the contrary, a significant acute toxicity for Daphnia as well as a clear chronic toxicity for luminescent bacteria were detected [110]. Again, the need for application of multiple bioassays is evident. Information useful to design sustainable, alternative, solvents can be obtained performing detailed biological studies. Methyl- and some ethylimidazolium ionic liquids have been tested on luminescent bacteria as well as on the IPC-81 (leukaemia cells) and C6 (glioma cells) rat cell lines [111]. The Effect Concentrations were generally some orders of magnitude lower than for acetone, acetonitrile, methanol and methyl-tert-buthylether, pointing out and quantifying the clear influence of the alkyl chain length on toxicity. The effects of pentachlorophenol [112] have been studied using a battery of eco-toxicological model systems: immobilization of D. magna, bioluminescence inhibition in the bacterium V. fischeri, growth inhibition of the alga Chlorella vulgaris, micronuclei induction in the plant Allium cepa, inhibition of cell proliferation and MTT reduction in Vero cells, neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity in the salmonid fish cell line RTG-2. The sensitivity of the different system resulted: micronuclei induction in A. cepa >D. Magna immobilization > bioluminescence inhibition at 60 min > cell proliferation inhibition of RTG-2 cells. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity [113]. The classic applications of luminescent assays, i.e. the assessment of the toxicity in polluted environments or the monitoring of remediation processes, are still of great topical interest. Environmental hazard of sites contaminated by coal tar and its product, is usually ascribed to pollutants such as the 16 polycyclic aromatic hydrocarbons (PAHs) prioritized by the U.S. Environmental Protection Agency (U.S. EPA). Such hazard was evaluated by the Lumistox test, a luminescent bacteria commercial kit. Pure naphthalene, acenaphthylene, acenaphthene, fluorene, and phenanthrene revealed inhibiting effects, but elutriates of PAH-contaminated soils produced a negligible inhibition of the



light emission, since the amount of PAHs was very low. In this case, the solubility problems impaired the BLB test applicability. Lumistox 300 has been again employed to test the toxicity of 11 reactive dyestuffs and 8 auxiliaries from a textile dyeing and finishing mill [113], as well as of the degradation products of azo reactive dyes treated by photo-Fenton and Fenton-like oxidation reactions [114]. In both cases, the results demonstrated that the toxicity assessment with luminescent bacteria is effective and of practical use, with the only exception of samples from deep dark-coloured dye bath samples and from the related effluents.

Applications in Detecting Inorganic Pollutants

The assessment of mercury toxicity was performed comparing three microbial test systems: the Microtox, the motility test by using Spirillum volutans, and the growth zone inhibition test on B. cereus. Out of these three tests, Microtox was found to be the most sensitive [115]. Again, Microtox test was used to assess the toxicity of the solid and dissolved corrosion products of galvanostatic polarization: Co-Cr-Mo corrosion products were found to be more toxic than those of stainless steel, and the most toxic ions were Cr(VI), Ni2+, and Co2+ [116]. The biological effects of Arsenic, in solution under the arsenate or arsenite forms-As(V) and As(III) [117] have been also investigated by the Microtox assay.

Future Applications of Bioluminescent organisms

It’s no more a fantasy; Bioluminescence in near future will serve to play a major role in different arena of life. At present only two companies engage in bioluminescent work; within a decade we foresee many companies coming up with the product of bioluminescence

Synthetic biology attempts to make biological systems more reliable, predictable and controllable. Naturally any biosensor application requires that luminescence is tightly controlled based on an input. For lighting applications too, we might only want luminescence at a particular time of day, or we might want our organisms to report on their environment glowing in response to pollution or perhaps offering a reminder to wear sunscreen when the UV index is high. Through regulating the luminescence gene networks, engineers could also create a multitude of programmable colours and patterns.

Luminescent reporters as used in current research generally modulate light output by changing the level of transcription of the luciferase gene. This creates a model that is slow to react to changes because the protein’s half life is on the order of hours. Some approaches have destabilised the protein in order to give more responsive regulation [118] but this approach still has limits.

Nature shows us that very tight control of luminescence is possible, with Japanese fireflies able to synchronise their ashes to within

milliseconds. This is made possible by the fact that the fireflies’ luciferase is localised to the peroxisomes. Nerve activity, relayed by nitric oxide signalling, causes ATP either to be consumed by the mitochondria or allowed to enter the peroxisomes to produce light. These sorts of approaches may be necessary to achieve luminescent light that is activated instantaneously as if by a light switch.

For lighting applications, a chassis would have limited resources to devote to luminescence. Brightness could be increased by restricting light output to certain tissues or to certain times of day. The clock genes in Arabidopsis are a model for circadian rhythms and are well characterised [119].

Glowing plants would only be visible at night anyway so it would be sensible to have luminescence genes controlled by a clock gene promoter. Plants also track the seasons perhaps flowering in spring and setting seed in autumn. They are able to achieve this by using photoreceptors to monitor the changing day length. By tapping into the gene networks that are modulated as a result, a gardener might be able to cultivate plants that only glow when they flower, or that are specifically designed to brighten up the dark winter months.

A common dream of future gazing synthetic biologists is a natural world displaying warnings of pollution, or operating as natural clocks. Biosensors are a key application in current synthetic biology and a research area that will encompass any detector module connected to any bioluminescent module to allow different light outputs to communicate changes occurring at a molecular level in a way that is easily visible.

Manipulating Vibrio Fischeri and Pyrocystis Fusiformis For Design Purposes

This work shows the analysis and results obtained manipulating two different kinds of glowing micro-organisms. They were grown to test their possibilities and build design devices that do not consume electricity to emit light. The work digs into the living swarm forms of intelligence they show, their living conditions, and the way they could be implemented in artificial geometries and structures for design purposes. It also presents some design proposals based on what was learnt. The two different species treated are Vibrio fischeri and Pyrocystis fusiformis. Light system for highways using Pyrocystis Fusiformis is already being worked out. Vibrio fischeri could be used for static commercial billboards. In that case, the bacteria would make the images more visible at night but the structure where the bacteria would be implemented should reproduce the image very clearly. Otherwise the image would not be visible. Eventually the image would disappear but that might be an advantage for a commercial strategy. It would also take a while for the image to be recognizable while the bacteria are growing.

Vibrio fischeri could also be used to create ambient lighting for interior or exterior spaces at night. It could be embedded in



modules able to be aggregated to form 3D usable structures. These structures would then emit faint light generating different ambient conditions around them. The bacteria could be placed in removable small containers attached to the modules so that they can be replaced or the modules could be filled with nutrients inside

Bioluminescent Immobilized Systems and Biosensors

Bacterial luciferase immobilized systems can offer a unique and general tool to analyse many chemicals and enzymes in the environment, exactly like they are already employed in other fields like research and clinical laboratories. When the luminescent reagents and enzymes, or the whole cells, are immobilized on solid supports their peculiarities changes, usually in a positive way. The sensitivity, specificity, and stability of the light-producing systems may be improved by using immobilized enzymes and they can be used for several analyses, reducing costs. The sensitivity is generally increased owing to the creation of a microenvironment with locally high concentrations of the involved reagents. Several solid supports and procedures are available to immobilize macromolecules or whole cells. The chemical methods give better yields, in terms of active luciferase, than the physical ones. Nevertheless, chemical procedures involve covalent coupling, and usually lead to some protein inactivation. Agarose, collagen, epoxy methacrylate and nylon have proved to be the most effective among the different solid supports that have been investigated. Gel entrapment technique has the advantage of better protein stability and enables the co-immobilization of luciferase and other enzymes with their substrates through an easy process [120]. An important, general, feature of these immobilized enzymes is the possibility to incorporate them into flow cells, used for multiple assays, recycled and reused in automated devices. The continuous flow format offers greater possibilities than a single-batch system, and it leads to rapid and sensitive assays. Flow assays are characterized by extremely accelerated kinetics: a very high surface-area-to-volume ratio is obtained and the reactions do not have to rely on passive diffusion to bring reagents together. Many analytes can be detected at pmol levels, with good precision and a wide range of linearity [121]. The immobilization of biological components on a solid support is quite regularly the preliminary step to the creation of a biosensor. In fact, biosensors are generally described as probe-type devices made up of a selective biological layer, with very sharp molecular-recognition capacity, and of a physicochemical transducer, often an optical system. Among these transducers, the (bio or chemi) luminescent ones have the advantages that they do not require light sources and monochromators [122]. Any kind of luminescent bacteria can undergo immobilization, including the genetically modified ones. A review, showing how the recombinant bioluminescent bacteria can be utilized as environmental biosensors is well elucidated by Gu [123]. Examples are reported

on phenanthrene toxicity in soil samples, of benzene in gaseous form. With further findings and developments of new non-specific stress promoters, the potency and extent of the information that can be obtained using these environmental biosensors is immense [123].

Environmental Applications

The bioluminescence intensity that reflects the overall health of the organisms and the luminescence reaction, which reflects metabolism, is sensitive to a wide variety of toxic substances. This sensitivity has made them a popular choice for methods to detect environmental pollutants, such as heavy metals and pesticides.

The presence of environmental contaminants such as organic and inorganic compounds, pesticides, and herbicides in runoff that feeds into drinking supplies is a major current health concern. For example, Atrazine causes mammary gland tumors if ingested at toxic levels. Copper, zinc, and nitrate compounds can cause gastrointestinal damage, especially in fragile infants, who may die if continuously exposed. Thus, a proper method of determining the presence of such contaminants is crucial to improving water quality and public health.

An economical bioassay method for determining the toxicity of aquatic contaminants in developing countries should help improve public health worldwide. This research aims to explore the effect of different organic and inorganic contaminants on the bioluminescence of the bacterium.

The reduction in bioluminescence intensity of cultures grown in liquid flask or solid plate cultures can be correlated with the amount of toxicity from environmental contaminants such as ZnSO4, CuSO4, NaNO3, HgCl2 etc. [124] with the use of luminometer that will quantify the data in the form of relative light units (RLU).

Bacterial sensor, which consist of living micro-organisms or genetically engineered microbes to produce specific output in response to target chemicals, offer an interesting alternative to monitoring approaches. Bacterial sensor-reporters detect bioavailable and/or bioaccessible compound fractions in samples. Currently, a variety of environmental pollutants can be targeted by specific biosensor-reporters. Although most of such strains are still confined to the lab, several recent reports have demonstrated utility of bacterial sensing-reporting in the field, with method detection limits in the nano molar range. This work seeks to understand the general design principles for bacterial sensor-reporters, thus presenting an overview of the existing biosensor-reporter strains with emphasis on organic and inorganic compound detection. A specific focus throughout is on the concepts of bioavailability and bioaccessibility, and how bioluminescent bacteria-based sensing-systems can help to improve our basic understanding of the different processes at work.



Water

samples represent the easier environmental matrix to be tested by bioluminescence assays, which, for the main part, are based on marine bacteria. To apply these assays in the assessment of toxicants has a great importance for the wellness and effective protection of ecosystems and of human health. New researches are continuously carried out on this topic, a large number of them concerning the control of wastewaters and treatment plants efficiency. The toxicity of the influents from industries into urban wastewaters, of the effluents from treatment plants, as well as of the water in the various compartments, must be continuously monitored.

This is necessary to avoid damages to the activated sludge, to check, at any moment, the effectiveness of the treatments and the quality of water released to the environment. The sensitivity, selectivity and robustness required to the bioassays in the mentioned situations are greatly different, and then several organisms, often isolated from this special environment and genetically engineered, have been tested and developed to comply with the needs of extreme conditions. Studies employing BLB assays in wastewater monitoring will be reviewed as first, and then the applications to the various aquatic environments will follow.

Tap Water

The interest in online drinking water quality monitoring has increased significantly in the last years. The quality control methods for water intended for human uses must be the more sensitive, reliable and complete among all those available in this field. The aim is to ensure the consumers, and the control authorities, that any compound with possible adverse effects on human health is absent, or under the safety levels. This lead to the introduction of new monitors, which can provide (near) real-time information on water quality, for example in continuous river water quality control as well as in drinking water protection against intentional contamination. The combination of complementary assays, like a chemical analytical technique and a bioassay, into a single integrated monitoring platform, would greatly enhance the performance of the control systems. Where a chemical analysis identifies and quantifies specific contaminants, biomonitoring gives an indication of the total quality, including the effects of unknown, toxic substances. Such a combination has been realized in the TOX control, a biological toxicity monitor, which include luminescent bacteria and a submersible UV-vis spectrophotometer probe, the scan spectroyser TM. It has been applied in the evaluation of drinking water safety. The alarm signals from one instrument can be verified with the signal from the other, reducing false alarm rates [125]. Another combined system has been used to evaluate the effectiveness of innovative drinking-water treatments, designed to remove toxic and mutagenic organic micro pollutants

from lake waters used for human consumption. Lake water samples were analyzed for mutagenic activity using Ames assay, for toxicity using bioluminescent bacteria, and for the presence of organic compounds using the GC-MS technique in research studies [126].

Feasibility of Bio-Lighting

Most current applications of bioluminescence in synthetic biology emit light barely visible to the naked eye, thus requiring sensitive electronic or chemical detection. Therefore optimization in research process might one day use bioluminescence reaction for lighting our lives. Many studies in future aims at feasibility of bioluminescence lighting, particularly whether or not a photosynthetic organism could derive enough energy from sunlight to give useful light output [55]. A detailed but by no means comprehensive analysis is provided by research studies with particular focus on estimating whether a large luminescent tree could compete with a fluorescent streetlight.

Communication With Human

Since the lighting of the flaming beacons that formed the first lighthouses, light has been an important medium for communicating information. It is now ubiquitous in this role, from the red, yellow and green of traffic lights and crossing signs to the technicolor output of television screens and projectors.

Wherever a biosensor is used, whether to measure the levels of contamination in ground water or the glucose in a diabetic person’s blood, some mechanism is needed by which the molecular interactions that measure the target molecule can be turned into a human read able output. Today this often involves an electronic intermediary, but here is the possibility that these systems might one day be entirely biological, with bioluminescent light replacing the status Light Emitting Diode (LEDs that exist today.

Nanoscience in Bioluminescence

Fireflies

The efficiency and lambency of firefly bioluminescence has long attracted the curiosity of scientists in Nanotechnology. This curiosity, coupled with advances in ‘quantum nanorods’, paved the way for a team of researchers from Syracuse University to conduct a full scale experiment manipulating the interface between the biological and non biological components [121].

In fireflies bioluminescence glow is expressed at nano level by exciting electrons by an enzyme. And when exited electrons de-exites it becomes dark.

The researchers discovered a way to harness the bioluminescence of fireflies by using the nanorods to manually introduce fireflies naturally occurring enzymes. More specifically the scientist simulated the chemical reaction fireflies create by combining



luciferin to the enzyme luciferase. The resulting glow is called Bioluminescence Resonance Energy transfer (BRET).

Hawaiian Bobtail Squid

Another use of nanoscale activity is exhibited by Hawaiian Bobtail Squid, which saves itself from predators in night by producing luminescent light at nano scale from the underside, where it contains bioluminescent bacteria. Moreover, it has stacks of silvery nano-plates behind the tissue, which reflects light down wards and prevents it from casting a shadow.

And finally a red bioluminescence producing glow at the nano-scale called Siphonophore a jelly fish type invertebrate which produces the glow to attack a prey.

Quantum Dots

Researchers focused beyond nano and entered in the domain of quantum dots and its relation to glowing technology. The quantum nanorods- which contain an inner nucleus of cadmium selenide surrounded by an outer shell of cadmium sulphide- are being created with the success of the experiment, as well as the ability to produce colour variations not possible with normal fireflies. The findings could lead to consumer products that are lit by multicolour nodes of light that don’t need batteries or electricity. The long term hope is that this nanorod technology could be used to replace LEDs and generate bioluminescent, renewable, energy less lighting for products and possibly homes and businesses too.

BRET quantum dot nano-particle has also been used in self illuminating in vivo lymphatic imaging in mice [120]. It allows performing real time, quantitative lymphatic imaging without image processing. BRET-Q-dots have the potential to be a robust nano-material platform for developing optical molecular imaging probes.

Entry of Bioluminescence in Synthetic Biology and Some Concerns

The young field of synthetic biology has dabbled with bioluminescence and it looks set to be a part of many emerging technologies in this area, both in the near and more distant future. Synthetic biology aims to design and model novel biomolecular components, networks and pathways. These are then applied to rewire and reprogram organisms to provide solutions for various challenges [122]. Engineered biological systems plan to utilise light emission for a variety of purposes. Proposed applications cover many areas of human activity, from bioluminescent trees for lighting to the use of light for cell to cell communication and each will require differently optimised bioluminescent systems.

To the synthetic biologist take luminescent microbes as an archive from which enzymes with desired properties can be selected. The

engineer must then aim for a synergy between the bioluminescent parts, other genetic elements drawn from other parts of nature, and the biological `chassis’ in which they are all housed.

Sometimes natural parts may need to be reengineered to better suit the purpose for which the organism is intended. The designer must also consider the resultant genetically modified organism, and any risk it might pose to the environment. Where there are risks, it may be possible to mitigate these by building in biological and genetic containment systems. Synthetic biology utilizes modularisation; which intends to allow any bioluminescent module to be used in a wide variety of contexts. The use of different promoters for luminescence could be induced by different environmental conditions; or through more complex approaches tight spatial and temporal control. Through characterisation and modelling, synthetic biologists work to optimise and improve biological systems. Experiments so far have showed promise, light output has been low but proofs of concept have been demonstrated and key areas for improvement highlighted. Engineered bioluminescence has a very exciting future, fuelled by increased understanding knowledge of natural biochemistries and improvised technologies driving synthetic biology,

Novel methods and technology have enhanced understanding of the molecular basis of bioluminescence, its physiological control, and its significance in marine communities. New methodology derived from understanding the biochemistry of natural light-producing molecules have led to countless valuable applications. To understand these interactions and the distributions of luminous organisms, recently developed instruments and platforms allow observations on individual to oceanographic scales.

Conclusion

From the data reported in the wide collection of papers composing this review, it is possible to conclude that BLB tests are, on average, enough sensitive to detect compounds that can be toxic to humans and to the whole environment. The continuous development, and application, of bioluminescent alarm-tests support this assertion. The sensitivity, ease of use, rapidity, flexibility and low costs of the Bioluminescence test systems, when compared to the features of other bioassays, suggest luminescent assays as the better choice, at least concerning some of the cited characteristics. Genetic manipulation techniques, transforming in luminescence emitters the native bacteria from the environmental matrix to analyse, increased further the applicability and reliability of Genetically Modified Bioluminescence Bacteria (GM BLB) assays, many of them cited in this review.

The final decision about which kind of method, chemical, biological, or a combination, is better to apply in each specific situation will depends on the most important features required in that case.



For example, in developing countries two of the most important parameters to take into account are the technical possibilities of the local institutions, and the economic impact of the project, depending also on the starting situation and on the goals to reach. Any case, and once again, the principle that to monitor effectively an ecosystem a battery of bioassays, luminescent or not, is necessary has to be always followed. The combination to chemical analysis can be fundamental in some cases, very useful in others. Even in the case that only luminescence-based assays are employed, several tests must be used simultaneously, as they differ in their sensitivity to diverse contaminants. A list of papers reporting the contemporary application of diverse bioassays is reported.

Natural bioluminescence is wondrous and efficient, and likely to be a key part of biological engineering in the future. Exciting proposals range from the strictly practical to the deliberately beautiful. Bioluminescence is a feasible source of useful illumination, though it will never outcompete conventional lighting in terms of sheer brightness. Instead it seems likely to complement existing light sources, offering a more aesthetic alter native or conveying useful information. There are many challenges along the way. Our knowledge of natural bioluminescent systems and of synthetic biology strategies to transfer and optimise them is limited but ever increasing. We must of course be responsible with the design and implementation of new technologies and consider their wider impacts. Care must be taken to identify and minimise any risks. A synthetic biology approach values high modularity and well-characterised parts, so adaptable bioluminescent systems could be produced and optimised for a range of applications. Luminescent biosensing and communication applications are still in their infancy and biolighting only theoretical at this stage but early studies have proved the principles and high-lighted the areas of improvement needed. With our knowledge and techniques ever improving, increasingly amazing bioluminescent systems and organisms will be available to scientists and consumers. These developments could herald a bright and exciting future.

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