A Novel Strategy: Microbes in Relation to Heavy Metals Toxicity and Future Prospects

The heavy metals persistence in the environment may pollute or contaminate aqueous streams and soils. In this respect, an option of bioremediation process offers to take into account the microbes and plants to reduce the possible harmful effect or to destroy the various contaminants. Under the metal stress, numerous strategies have been developed to avoid the heavy metal toxicity by the soil microbes. Plants growth significantly improves when these metal resistant microbes used in soils as bio-fertilizers or bio-inoculant that are stressed by the heavy metal. It proves to be a highly environmental friendly, easy and cost-effective approach to apply the beneficial plant traits along with the microbes that have efficient properties of heavy metal detoxification. Bioremediation by microbes and phyto-remediation between the various processes of bioremediation are quite operative. Some microbes allow the desired process optimization through varying physicochemical conditions of the contaminated area by catalyzing the heavy metal related reactions. The combination of microbial community genetic engineering will prove to be paramount importance for polluted sites. This review summaries the key significant properties and phenotypes of the microbes that could be contributory to heavy metal bioremediation, along with their biochemical background and community genetic engineering.


Maize (Zea mays)
Reduction in seed germination; decrease in plant nutrient content; reduced shoot and root length Reduced shoot growth; inhibition of root growth [12,13] Co Mung bean (Vigna radiata) Tomato (Lycopersicon esculentum) Reduction in antioxidant enzyme activities; decrease in plant sugar, starch, amino acids, and protein content Reduction in plant nutrient content [14,15] Cr Wheat (Triticum sp.) Tomato (Lycopersicon esculentum) Reduced shoot and root growth The decrease in plant nutrient acquisition [16,17] Cu Bean (Phaseolus vulgaris) Accumulation of Cu in plant roots; root malformation and reduction [18] Hg Tomato (Lycopersicon esculentum) Rice (Oryza sativa) Reduction in germination percentage; reduced plant height; reduction in flowering and fruit weight; chlorosis The decrease in plant height; reduced tiller and panicle formation; yield reduction; bioaccumulation in shoot and root of seedlings [19,20] Mn Broad bean (Vicia faba) Mn accumulation shoot and root; reduction in shoot and root length; chlorosis [21] Ni Rice (Oryza sativa) Wheat (Triticum sp.) Inhibition of root growth Reduction in plant nutrient acquisition [22,23] Pb Maize (Zea mays) Reduction in germination percentage; suppressed growth; reduced plant biomass; decrease in plant protein content [24]

Introduction
Heavy metal contaminants are the most important concern in water and soils due to persistence and toxicity [1,2]. The toxic level of heavy metals is 1.0-10 mg/L. Remarkably: even though at a very low concentration level 0.001-0.1 mg/L, metal ion of Cd and Hg show toxicity. Under some conditions, it is also possible that some heavy metals may also be transformed from less toxic to high toxic (e.g., Hg). Plant tissues can absorb heavy metals from metal contaminated soil which affect the plant performance ( Table 1) that may also accumulate in the food web up to trophic level [3,4] and so their poisoning may result in intra-uterine retardation, irreparable nerve, tumors and organ damage in humans. Some toxic symptoms may appear after a long time of exposure [5]. Regardless of heavy metal dangerous toxic levels, in many world parts, it is increasing day by day [6][7][8][9].
Recently the attention of microbiologists is more focused on specific plant-associated microbes [25]. Soil microorganisms under metal stress, including PGPB have developed many strategies to avoid different heavy metals toxicity. As soil is a very complex ecosystem in relation to physiochemical and biological components [26][27][28][29][30], so to maintain the productivity of plants [31,32] and fertility of soil, vital role played by various microbes can be considered [33,34]. These mechanisms may include bioaccumulation, the adsorption of metals on the cell wall, the removal of metal and biotransformation outside the microbial cell surface [35].
For a better understanding of microbes living in heavy metal contaminated soil, their mechanism of growth-promoting with these contaminants to cope with their natural capacity could be exploited for sustainable crop growth; industries feed stock, the production of bio-fuel and for the biomass on the metal-contaminated land [36]. Furthermore, phytoremediation processes efficiency could be efficiently improved by introducing and exploiting the highly efficient plant-associated microbes [37,38].
Bioremediation is an opportunity that provides the biological activity for natural destruction of contaminants or extracts them safely. Bioremediation is a natural process which depends on bacteria and fungi, as these microorganisms carry out normal life functions to change these contaminants by heavy metal immobilization as source of energy on the surface of the cell, interpreting these to less toxic or harmless products by precipitation and oxidation-reduction acidification in most cases [39]. Thus, an alternative tool is provided by bioremediation through biological activity to render or destroy the harmful . 03 .
Microbial degradation has been projected for the remediation of heavy metal toxicity as it is a highly efficient strategy than other Physico-chemical remediation methods [41,42]. It has distinctive advantages; as being low cost, to achieve the desired goals may be conceded out on site without site disturbance of native fauna, flora and it is also low technology technique.
This article surveys the variety of bacterial capacities to cope with heavy metal toxic concentrations measured generally to be environmental contaminants and in co-contaminated environments strategies for improvement efficiently aimed at increasing biodegradation. In certain strains, valuable properties present can be upgraded or combined by genetic engineering. This review will not only concentrate on the efficacy and degree of phytoextraction focuses to improve trace element uptake by plants that how plant-associated microbes can contribute in reduction of heavy metal toxicity but also give a deep insight on those amenable to genetic investigation of microbial systems and ultimately it will lead for the enhancement of their metal-associated abilities to microbial community-engineering strategies and genetic engineering.

Heavy metal toxicity resistant strategies adopted by microbes and metal resistant microbes
Microbes have developed some strategies to astound the inhibitory effects of heavy metals toxicity. Such metal detoxification strategies adopted by microbial communities are; far away from the cell, the active transport of metals extracellular sequestration, by protein binding, metal intracellular sequestration, enzymatic detoxification of metal, decreasing the metal ions sensitivity to cellular targets and by permeable barriers of metal exclusion. There are three possible mechanisms by which these systems operate ( Figure 1). First, the specific ion accumulation can be reduced by efflux, that prevents the heavy metal entry into the cell [43], that include the members of the RND (Resistance-Nodulation-Cell Division) protein family, CDF family (Cation Diffusion Facilitators), against heavy metal cations P-type ATPases-basic, CHR protein family, export superfluous cations, secondary cation filters, defense CnrT and NreB. Second, the sulfur lovers, cations, can be separated by molecules containing thiol, particularly into complex compounds. Third, some metal ions may reduce to a low toxic oxidation state.
Dual bio-augmentation comprising inoculation together with organic-degrading and metal-detoxifying bacteria within a co-contaminated system was investigated to facilitate organic degradation [44]. 500 μg of 2,4-D/mL amended in uncontaminated sandy loam soil, soil microcosms were constructed with Cd 60 μg/mL of final concentration. This was followed by inoculation with a Pseudomonas H1, a cadmiumresistant strain. It was concluded that dual bio-augmentation for remediation with microbial populations organic-degrading and metal-detoxifying is efficient for co-contaminated soil remediation; though preceding to inoculation with reducing metal concentrations bioavailability via sequestration will raise increased degradation with the organic-degrading population. So, the eventual choice for bio-augmentation of gene or cell will depend on the potential relative health of the recipient population, the time frame available for remediation and the degree of contamination  [58] ( Table 2). The strain was revealed to produce IAA, which might be responsible for the promotion of plant growth. Similarly, a Pseudomonas strain that is Cr 6+ resistant was able to stimulate B. juncea growth by producing IAA and thus improved the extraction of trace elements [59]. Bacillus sp. strain, those haven't produce IAA, boost Cr 6+ extraction and upsurge plant growth though to a lesser extent. Biomass stimulation of B. coddii in mycorrhizal plants led to a higher Ni total content [60]. Whiting et al., compared to Zn accumulation in Thlaspi caerulescens (hyperaccumulator) and Thlaspi arvense (nonaccumulator) by rhizosphere bacteria [61]. The authors showed that Zn uptake in Thlaspi caerulescens assisted by rhizosphere microflora, while T. arvense remained unaffected. Interestingly, the bacterial number was high in T. caerulescens rhizosphere than T. arvense [61]. In both, soil-grown and hydroponically plants, inoculating the S. alfredii with trace element-tolerant rhizobacterial strains belongs to Burkholderia genera that enhanced the plant metal tolerance, Zn, Cd extraction, uptake, and biomass production [62].
For instance, hyperaccumulating rhizosphere bacteria initially isolated from plants have shown to stimulate growth and development of diverse plant species [57,132]. Psychrobacter sp. SRS8 Ni resistant PGPB strain isolated originated from Ni-hyperaccumulator rhizosphere stimulate phytoextraction potential and growth of crops. Becerra-Castro et al., assessed the fourteen Cd and Zn resistant bacterial strains of in rhizosphere that effect on Salix viminalis and Festuca pratensis [133]. Almost all strains stimulated growth, the most strains showed a negative influence on Salix and the strains Massilia sp., Rhodococcus sp. Pseudomonas sp. and Streptomyces sp. showed a positive effect regarding the heavy metal detoxification. In contrast, [61] demonstrated that rhizosphere micro flora assisted Zn uptake and biomass production of the T. caerulescens hyperaccumulator, while the T. arvense non-accumulator did not affect the uptake of Zn. These studies highpoint the complex nature of plantmicrobial interactions and the requisite to optimize the plantmicrobial system. However, given the rhizosphere, diversity of soil and endophytic trace element tolerant microorganisms are the openings to discover the beneficial plant-microbial partnerships.
As enlightened above, microorganisms have adjusting mechanisms including PGPB to heavy metal contaminants that are unceasingly exposed to these metal stresses [134]. Microbes respond by various biological processes to these molecules like precipitation, transportation across the cell membrane. The bacterial response to a particular heavy metal for exploiting them is of great importance in metal-contaminated site remediation [135]. Even though PGPB has been used in agronomic practices as growth-stimulating agents, substantial emphasis has been given to exploit the heavy metal detoxifying potential for the phytoremediation (including phytostabilization . 05 .

Improving the process of phytoextraction through community engineering
The prospective for using native communities of soil microbes has yet to be entirely explored to boost phytoextraction. Just as uranium-contaminated groundwater, in situ bioremediation makes use of metabolic processes and ubiquitous microorganisms, community engineering might be used for the selection of indigenous microorganisms that mobilize heavy metals or encourage plant growth in soils [138]. The in situ-microbial communities use for remediation in soil have the supreme beneficial as firstly, using communities in situ refutes the concern of the performance of variable inoculum in altered soil conditions through using from every given soil, the best plant growth promoters. Secondly indoor community competition can be utilized as leverage to increase the numbers of microorganisms that are assisting phytoextraction instead of a hurdle impeding the retention and colonization of an inoculum. Thus, in situ-microbial communities can be exploited by stimulation of bacteria to increase phytoextraction that assists plant-uptake by mobilization of heavy metals. The reversible nature of immobilization and mobilization reactions that could limit in situ remediations of groundwater is less likely to inhibit the phytoextraction since heavy metals are physically removed from the soil. Actually, the combination of the successional immobilization and mobilization of Cd phytoextraction through Geobacter sp. Cd1 has been proposed due to the above-mentioned reason [139].
Besides microbial redox reactions, phytoextraction improved by community engineering can target the known PGP promotion of the genre (For instance, the Bacillus, Rhizobium, Enterobacter, and Pseudomonas) [140]. Though, the conditions in the existence of heavy metals which favor the proliferation of induced activities of PGP or these microbes haven't been clearly probed.
Microbial PGP stimulation is an alternating approach of community engineering which targets the biochemical processes that supposed to be connected for the promotion of plant growth ( Table 2). The IAA produced by microorganisms could increase phytoextraction [141]. Though there are multiple pathways of IAA synthesis, Indole-3-acetamide, Tryptamin, Indole-3-pyruvate, Indole-3-acetonitrile, and Tryptophan sidechain oxidase pathway, these converge on precursor tryptophan. The in vitro tryptophan addition can stimulate the production of microbial IAA and for up-regulating IAA pathways in situ substrates can be used [142]. For community engineering, the other microbial processes that can target fermentative processes that yield carboxylic acid short-chain for instance oxalic acid, citric acid, tartaric acid and acetic acid are assisted by PSMs [143]. Sugars like beet molasses and glucose, which are used for lactic and critic acid industrial production respectively, are the potential preliminary points for stimulating the organic acids production by PSMs.
Community engineering targeted processes will be supreme active in the rhizosphere. From bulk soil, rhizosphere communities assembly is believed to be a principally deterministic process and evidence is there that microorganisms for certain soil are enriched further in the rhizosphere of the plant [144]. Such as, in hydrocarbons presence, microorganisms have selectively advantaged in the soil that are capable of their degradation. Though, microorganisms that encode the hydrocarbon metabolizing genes comprise of xylE, alkB and ndoB have revealed that, in contamination presence, hydrocarbondegrading ecotypes enrichment is larger in rhizosphere relative to the bulk soils [145]. Therefore, soil communities engineering . 08 .
to encourage a definite microbial process will enterprise these processes enrichment in the rhizosphere having the supreme influence on phytoextraction.

Technological advancement regarding microbial community engineering
The technologies that assist the strategies of microbial community engineering to enhance the bioremediation are fetching progressively refined and manageable. Profiling 16S rRNA can be used for the community to describe taxonomically [146,147]. Profiling 16S rRNA can track the key genre response to the soil treatments and identify strategies of community engineering that enhance and stimulate their growth.
The high-throughput sequencing is highly difficult that is used to investigate metabolic processes and biochemical pathways of concern. Shotgun meta-genomics allows entire genomes reconstruction from multiple organisms. Yet, in soils high diversity makes it challenging to create adequate of rarer readcoverage genomes without considerable sequencing amounts [148]. Hence, usually, only the most abundant community member's genomes are able to be amassed.
Metagenome sequencing approaches to predict communities functional potential. Predictive metagenomics software (tax4fun and PICRUSt), help in amplicon-based strength and by associating the sequences of 16S rRNA taxonomic information to prevailing assembled genomes databases [149]. In community engineering, predictive meta-genomics might be used as a primary strategy to investigate whether changes to taxonomic profiles are translating the changes in the functional potential of communities. Alternatively, like Geo Chip microarray technologies could be used in combination with community mRNA or DNA to track the changes in community functional activity [150]. Geo Chip microarrays capture information on main bio-geochemical cycles, comprising C, N, P, S, and numerous metals along with information on degradation of organic contaminant, antibiotic resistance, energy production and stress responses [150]. Geo Chip could be used to see the changes in the expression of mRNA of genes extracted from communities that are environmentally relevant to inform studies of community engineering. The microarray probes pathways of biosynthesis target genes allied with growth promotion of plant, like siderophore (entABCDEF) or IAA (iaaM and ipdC) biosynthesis pathways, will further enhance the improvement in the Geo Chip value and community engineering phytoextraction studies [142].

Microbial bio sorbents genetic engineering
Genetic engineering can be adopted in microbe aided remediation of heavy metal polluted soils. For instance, [151] described that in polluted soils, genetically engineered Ralstonia eutropha could be used for sequestering the metals. Even though sequestered metals remain in the soil, less harmful as they become less bio available.
In genetic engineering first studies of bio-sorbents involved eukaryotic MTs cloning in bacteria for their intracellular expression. MTs are of low molecular mass proteins family that is able to coordinate a variety of ions of heavy metal. They are being thought to establish the key mechanism by which eukaryotes guard themselves against these pollutants [152]. Human MT cytoplasmic production fused to araB in Escherichia Coli carried about a 3-5-fold increase in bioaccumulation of Cu and Cd [153]. Additionally, MT chelating efficiency proved to be greater when directed to periplasmic space [154]. As a result of this observation, and in order to evade the complications related with cytoplasmic expression i.e., limitations of metal uptake, interference with cytosol redox state and associated toxicity with an accumulation of intracellular metal, well ahead research directed MTs to E. coli outer membrane compartments or to periplasmic space [155]. A strain of E. coli expressing MT, merged to LamB (outer membrane maltose protein) indicated an increase of about 15-20-fold in Cd 2+ binding as compared to wild type equivalent [156]. A most effective alternative to heavy metal coordinating moieties surface display is cytoplasmic expression joint with particular metal membrane transporters introduction [157]. Across the cell membrane, this method overwhelms the limitations of metal uptake, but it is restricted for those heavy metals having active import systems (like Hg, Cu, Pb and Ni etc.). Likewise, bacteria that are engineered genetically co-expressing MerP-MerT Hg transporter with heavy metal-binding peptides or MTs in cytoplasm exhibited bioaccumulation of Hg comparable to cells that are directly expressing binding peptides on the surface of the cell [158]. At decreasing concentrations, system was proficient in effectively recovering and removing Hg 2+ [159]. An additional advantage of this approach is that heavy metal binding polypeptides cytoplasmic expression is also an operative system for the cellular detoxification of some metals [160]. There are only few reports that shows the a heavy metal biosorption in Gram-negative surface structures of bacteria through lipopolysaccharides.

Engineering phosphate and sulfide precipitation to aid the bioremediation
Heavy metal precipitation is mediated in phosphate precipitation engineering process by inorganic phosphate liberation from donor molecules of organic phosphate. Greatest work has conceded with Citrobacter sp. strain that is sequestered from heavy metal-polluted soil. It was exposed that this bacterium can mount up high levels of nickel and uranium through the formation of highly insoluble metal phosphates opening the way toward future applications in the bioremediation [161,162]. This phenomenon can be considered as an example of heavy metal biosorption, with bacterial metal phosphate metabolism formation that plays a vital role [41]. It has been proven feasible that transference of precipitation activity of metal phosphate from Citrobacter sp. to other bacteria. Enhanced biomineralization of metal phosphate in E. coli was achieved by the introduction of phoN from Citrobacter or of associated phosphatases [163].
Another tool to counteract the heavy metal is bacterial generation is the pathways of engineered polyphosphate. On the ostensible relationship this probability is based on the resistance of metal and polyphosphate reserves depletion in cells [164]. Into P. aeruginosa the inducible polyphosphate kinase introduction led to more quantities of polyphosphate accumulation. Under the conditions of phosphate limiting, if the organism is successively exposed to uranyl, polyphosphate will be degraded and the inorganic phosphate secreted, removing uranyl from solution [165].
Natural mechanism of metal precipitation is sulfide production by SRB. In the immobilization of metal sulfide in anaerobic sediments, SRB play critical role that contains high metals concentrations [166]. By this phenomenon in the continuous culture grown bacterial biofilms examining can be attributed to metal sulfides deposition at surface of biofilm or in liquid phase, followed by entrapment of the precipitated sulfides through exopolymer. In the treatment of leachates and waters SRB has successfully been used in bioreactors of big scale and at laboratory surveys used in pilot. Pure cultures bacterial consortia mixed with sulfate reducing are highly operative in elimination of metals from the solution [167]. By precipitation 98% bioleached metals can then be removed from effluent by using SRB containing anaerobic reactor. The SRB and bacteria that are iron reducing like Geobacter and Shewanella species in heavy metals natural sediments places the base for their usage in processes of in situ remediations [166,168]. But, the fact even little levels 20-200 μM of free Cd(II), Ni(II) or Zn(II) ions proves to be toxic fr SRB such as their use may be limited by Desulfovibrio. In high concentrations of Cd 2+ Klebsiella planticola strain capable to thrive and precipitate significant amounts of cadmium sulfide that might be an alternative for immobilization of metal to SRB in the anaerobic conditions [169]. Furthermore, 98% available Cd from upto 200 mM solutions, removed by one of the recombinant strains under anaerobiosis [170,171].

Conclusion
Bioremediation with microbes is a rising technology that seems to resolve issues related to heavy metals toxicity without any lethal effects by converting heavy into bioavailable and soluble form, which facilitates ultimately their remediation. The biological approaches advantages include more high specificity than chemical and physical methods and their in situ suitability methodologies and potential for improvement by the genetic engineering. Genes can be selected that are specific to biodegradation and plant species in accordance to plant growth conditions and contaminants present at toxic sites. In specific, application of plant associated microbes that are engineered genetically can be a auspicious approach in phytoextraction improving procedures. Recent advances in software and sequencing technologies now allow the in deep research into community engineering of the in situ communities of microbes to boost up the phytoextraction of the heavy metals in the soil. By plants, uptake of heavy metals is directly enhanced by siderophores, redox processes biosurfactants, organic acids and biomethylation. In situ microbial communities' use supports that bioremediation can be improved efficiently by utilization of the microbes that are optimally adjusted to contaminated rhizosphere soil conditions. This opportunity of research will also greatly contribute to our soil-microbial ecological fundamental understanding and suggest to investigate this open research area to develop and construct new methods and techniques for toxic contaminants remediation.

Future Prospects
How to create a more vigorous system for heavy metal bioremediation brings new challenges to us. Some further prospects are mentioned that may open a new area of research. 1) Though more work is done related to microbial bioremediation of heavy metals but some key processes by the inoculated strains about colonization and interactions still requisite to be unknotted further in detail to allow microbial full scale application assisted contaminated soil bioremediation. 2) Least research is done to examine speciation changes of heavy metal in the rhizosphere. 3) As PGPR only can be colonize in certain plants, the scope of application is presently limited. 4) Heavy metal responsive genes, proteins, and metabolites genetic engineering have revealed astonishing results but complete prospective leftovers to be exploited. Use a different approach that is well integrated like transcriptomics, proteomics and metabolomics eventually requisite to meaningfully enhancing the heavy metal toxicity tolerance along with other abiotic stresses in plants that are important economically.
More research is required to compute the influence of processes of rhizosphere persuaded by rhizobacteria on heavy metals phytoavailability, involved mechanisms in metals transfer and mobilization so as to optimization of the process of phytoextraction and to develop future strategies. Such understanding can allow us to know their role and soil rhizobacteria mechanism on bioremediation.