A Review on the Resistance and Accumulation of Heavy Metals by Different Microbial Strains

Heavy metals accumulated the earth crust and causes extreme pollution. Accumulation of rich concentrations of heavy metals in environments can cause various human diseases which risks health and high ecological issues. Mercury, arsenic, lead, silver, cadmium, chromium, etc. are some heavy metals harmful to organisms at even very low concentration. Heavy metal pollution is increasing day by day due to industrialization, urbanization, mining, volcanic eruptions, weathering of rocks, etc. Different microbial strains have developed very efficient and unique mechanisms for tolerating heavy metals in polluted sites with eco-friendly techniques. Heavy metals are group of metals with density more than 5 g/cm3. Microorganisms are generally present in contaminated sites of heavy metals and they develop new strategies which are metabolism dependent or independent to tackle with the adverse effects of heavy metals. Bacteria, Algae, Fungi, Cyanobacteria uses in bioremediation technique and acts a biosorbent. Removal of heavy metal from contaminated sites using microbial strains is cheaper alternative. Mostly species involved in bioremediation include Enterobacter and Pseudomonas species and some of bacillus species too in bacteria. Aspergillus and Penicillin species used in heavy metal resistance in fungi. Various species of the brown algae and Cyanobacteria shows resistance in algae.


Introduction
Air, water and soil which are the essential elements of life are contaminated rapidly due to increasing population, urbanization, mining activities and industrialization [1]. Heavy metals toxicity is causing problem to humans, animals, aquatic animals, plants and even microbes too.
Various methods are introduced to remove the heavy metal pollution like chemical techniques such as chemical precipitation, oxidation or reduction method, electrochemical treatment. Physical techniques such as ion exchange, evaporation, filtration, membrane technology, reverse osmosis. Biological techniques like microorganisms such as bacteria, fungi, algae, cynobacteria, lichens, etc.
Heavy metals damage cell membranes, alter functioning of enzymes, inhibit protein synthesis, denature protein and damage the structure of DNA. Toxicity is mainly created by the dislocation of essential metals from their real binding sites or ligand interactions [2]. Bioremediation is cost-effective, safe and eco-friendly; can be virtually restored a result to the heavy metal pollution issue as it is natural process. Biological methods are best to control short term or long term environmental pollution. Various heavy metals are accumulated with the help of bacteria, fungi, cyanobacteria, lichens, etc. and helps in bioremediation and used as bioindicators. They are not harmful human heath as well as ecosystem. Such organisms are used for indication and controlling heavy metal pollution. Mostly genes encoded by heavy metal resistant bacteria are located on plasmids. Biosorption is environmentally safe and low cost methodology of removing metals from the ecosystem. Various analysis were observed throughout previous 5 decades provided quantity of data regarding differing kinds of biosorbents and their mechanism of absorption of heavy metal. Additional research is to explore new biosorbents from surroundings [3].
Since last few years, various physical and chemical methods are used to remove heavy metals but it is expensive, needs laboratory and inefficient. According to various studies bioremediation and biosorption techniques are much more beneficial, cheap, non-toxic, natural process.
Minimum inhibitory concentration (MIC) is the lowest concentration at which the isolate or antimicrobial agent is completely suppressed is recorded. Microorganisms correspond to heavy metals using various defense systems, such as exclusion, compartmentalization [4], complex formation and synthesis of binding proteins, such as metallothioneins [5].
Bioremediation strategies have been proposed as an attractive alternative owing to their low cost and high efficiency [6].
Different methods are used to study characterization of heavy metals on microbes by 16S RNA sequence, biodegradability test, siderophore assay, biochemical test, morphological test, antibiotic resistance, nucleotide sequencing, etc. Microbial pigmentation and enzymatic activities like catalase, gelatin hydrolysis, oxidase, nitrate reductase, were characteristics selected to examine their outcomes.
Bioremediation is of two types: in-situ bioremediation and ex-situ bioremediation. In-situ bioremediation process is mainly used due to its ability in decreasing disturbance of ecosystem at the heavy metal polluted sites whereas ex-situ bioremediation, it takes place inside bioreactors, bio-piles and land farming. In-situ bioremediation is much more efficient and eco-friendly (Figure 1).
Metal microbe interactions developed by microbial cells are bio-transformation, bio-leaching, bio-degradation, bio-mineralization, bio-adsorption and bio-accumulation in bioremediation method.
Biofilm used as efficient bioremediation tool and stabilization too. Even at harmful conditions, they show high resistance towards heavy metals. With the help of genetic engineering one can insert desired characters like ability to resist heavy metals, tolerate metal stress, etc. For example: engineered Chlamydomonas reinhardtii shows increased resistance to cadmium toxicity. Corynebacterium glutamicum was genetically modified using ars (operon) to accumulate arsenic polluted sites. Biofilm combines or work with biosorbent or any exopolymeric substance which consist of surfactants or emulsifier properties. The study was conducted on Rhodotorula mucilaginosa shows efficiency in heavy metal removal and develops 91.7-95.4% biofilm cells. Biosurfactants studied were surfactin, rhamnolipid and sophorolipid for removal of several heavy metals. application and burning of fossil fuels like coal, petroleum, etc. Chronic exposure of cadmium in human has many harmful effects such as high blood pressure and destroys to different organs such as lung, liver kidney and testes in males [18,19].

Copper in environment
Copper a transition metal and also an essential element for living organisms including humans and other animals at low concentrations. Copper is released in ecosystem through decaying of vegetation, forest fire, sea-sprays, wind-blown dust. Copper is utilized as the alloy in the manufacture of wire, pipe, and various metal products. Copper are majorly used in agriculture to treat plant diseases, like mildew, or for water treatment and as preservatives, leather and fabrics. Intake of excessive amount of copper, it can cause nausea, vomiting, stomach cramps, diarrhea and can destroy liver and kidney and even lead to death [20,21].

Zinc in environment
Zinc (Zn) is also a transition metal and zinc is utilized in galvanizing and alloying and also in the manufacture of electric goods, dying, insecticides, pesticides and cosmetics. Mining activities, smelting of metals and production of steel and other waste can release zinc into the environment. It may cause health issues in living organisms such as dehydration, nausea, electrolyte imbalance, vomiting, abdominal pain, dizziness, acute renal failure, muscular incardination and damage of hepatic parenchyma [22].

Manganese in environment
Manganese is released from sewage sludge, combustion of fossil fuels, mining processes, etc. it can cause toxicity in plants and causes swelling of cell walls, brown spots on leaves, etc. [23].

Iron in environment
The major sources of iron are metal refining, sewage, dust from iron mining, iron and steel industry. Iron sulphate is utilized in fertilizer and herbicide [24].

Other heavy metals in environment
Thallium is present in insecticides, metal alloys and fire cracker. Phosphorus is found in insecticides such as organophosphate for example: malathion [25].
The environmental factors plays very crucial role in biosorption of heavy metals and these factors are pH, temperature, biomass concentration, metal ion concentration. Algae, fungi and bacteria acts as biosorbents and helps in mechanism of biosorption [26].

Adverse effects of heavy metals
Heavy metal pollution is causing severe health effects in human body as well as animals and plants too. Heavy metals are also effected the growth of microbes which are used in treatment or accumulation of heavy metals by damaging their DNA. Heavy metals can cause skin allergies, cancer, effect major organs like kidney, liver, brain, lung, etc., and enter in blood stream and even death too in animals and Increased amount of zinc effects pancreas, disturbs the metabolism of protein and amino acids in body and arteriosclerosis too [33].
Cobalt can cause vomiting, nausea, loss of appetite and may affect on lungs causing asthma, pneumonia and wheezing when exposed with cobalt metal and may develop various allergies or skin rashes. Mainly it is dangerous for heart muscle and causes heart muscle disease known as cardiomyopathy and shows rapid increase in count of red blood cells after long time exposure [34].
Copper damages liver, brain, cornea, lungs, immune system including blood cells. It causes gastrointestinal symptoms such as vomiting, nausea, abdominal pain and even lead to liver and kidney damage, genetic disorders, reproductive or developmental effects, delayed growth, prolonged bone formation and less body weights [35].
Tin effect both nervous system and pulmonary system. Exposure may lead to skin and eye irritation or respiratory tract problems. It causes pneumoconiosis, central nervous system disorders, visual defects, changes in EEG too [36]. Phosphorus symptom caused by exposure of phosphorus on human health includes sweating, headache, vomiting, abdominal cramps, weakness, ptosis, miosis, and severe issues are sensorimotor, polyneuropathy, atrophy and even led to respiratory paralysis [37].
The consequences of thallium exposure include blood vomiting, nausea, abdomen pain, eye disorder, mental retardation, hair loss and severe issues are cardiac failure, brain disorder and even coma too [25].
Mercury attacks the nervous system and renal region and may cause proteinuria. Inhalation of mercury may cause headache, memory loss, insomnia, tremors, neuromuscular and thyroid damage. It damages the chromosome structure and DNA. Effects on reproductive system by low sperm count, birth defects and even miscarriages too. During pregnancy, it may pass through placental barrier to embryo or baby for exposure [38].
The major organs targeted by these heavy metal mercury and lead causes neurotoxicity (brain), arsenic lead to hepatotoxicity (liver), cadmium causes nephrotoxicity (kidney)/pulmonotoxicity (lungs) and zinc mainly induce hematoxicity (blood).
The heavy metals interrupt in metabolic processes in two ways [39]: • They are absorbed and thereby disturb role in major organs and glands such as the heart, liver, brain, kidneys, bone, etc.
• They displace the important nutritional minerals from their real place hindering their biological function. Consumption of foods, beverages, skin exposure, and the inhaled air are ways through which these contaminants can be present in body. It is unfeasible to reside in heavy metal free surrounding.
Various heavy metals produce ROS and damages DNA of the cell and disrupt reproduction cycle. Arsenic damages kidney and liver and may cause abdominal cramping, etc.

Adverse effects of heavy metals on marine animals
Heavy metals present in water by industrial effluent or agricultural waste like fertilizers, pesticides, etc. and deposited in water bodies and settle down and can present on surface with help of aquatic plants and aquatic macrophytes. Heavy metals stimulate the production of reactive oxygen species (ROS) that can damage aquatic organisms.
Several heavy metals accumulate in various major organs of the fish causing mortality. Firstly it affects the circulatory system by entering in blood and alters the components of blood. It makes the fish anemic and weak.
Huge amount of heavy metal shows inhibitory effects on the growth and development of aquatic organisms like fishes, phytoplankton and zooplankton. Heavy metals may cause disruption in respiration, damage respiratory track which leads to suffocation, reduces the sperm count, egg production and short life span. Heavy metals can disturb oxygen level, reduction of developmental growth or give rise to developmental anomalies, byssus formation and reproduction too. In juvenile phase shows high mortality and in adults decreased breeding ability. Heavy metal shows changes in structure and organs and may exhibit functional changes and transform metabolic pathways. Results of a research [40] showed that ten different fish species had the highest concentration of heavy metals is in liver and kidney.
The fishes like Labeo rohita and aquatic organism are eaten by humans as rich protein sources and heavy metal pollution may cause health risk in humans too through aquatic species. Cadmium can be bioaccumulated in mussels, oysters, shrimps, lobsters and fishes too.
Mercury in fish muscles occur as Methyl mercury which is formed in aquatic sediments. Movement of heavy metals in fish takes place through the blood where the ions are generally attached to proteins. There are five potential routes for the contaminants to enter an aquatic organism. The pathways are through the food, non-food particles, gills, the skin and oral consumption of water. Once the contaminants are accumulated, they are carried by the blood to the liver for modification and storage. If contaminants are altered by the liver, they can be stored or excreted in the bile produced in liver or reversed back into the blood stream for elimination by the gills or kidneys or stored in fat which is a hepatic tissue.

Adverse affects of heavy metals on plants
Plants require various heavy metals for their growth and excessive amount of heavy metals can damage cell structure, inhibition of major enzymes, inhibit the photosynthesis process and growth of plants, altered water balance, nutrient assimilation and can even cause plant death [41].
Heavy metal give rise to chlorosis, slow and poor plant growth, yield depression and even less nutrient absorption, disorders in plant metabolic processes and decreased potential to fixate molecular nitrogen in legumes of plants.
Seed germination was gradually retarded in the presence of large amount of lead. It can be due to long term incubation of the seeds and have resulted to compensate the toxic outcomes of lead by various mechanisms such as leaching, chelation, metal binding or absorption by microorganisms [42].
Replacing of major essential nutrients at cation exchange sites reveals indirect toxic effects on plant development. Enzyme metabolism is extremely crucial for growth and development of plants and heavy metals effect enzymes to inhibit many other major metabolisms in plants.
Heavy metals may lead to loss of fertility of soil by reduction in decomposition of organic matter by depletion of various microbes present inside the soil [43].
Copper is required as micronutrients in plants and helps in synthesis of ATP and assimilation of carbon dioxide. Excessive copper may exhibit oxidative stress and decreases growth of root.
Zinc required as micronutrient for synthesis of chlorophyll in plants. It retards growth of plants and nutrient level. It causes manganese and copper deficiency in shoot region. Cadmium results in inhibition of growth and development, browning of roots tips and even death too.
Mercury can effects whole food chain and induces ROS and oxidative stress too. It causes depletion of germination in seeds, height of plant reduced flowering and fruit production, retarded growth and development.
Chromium induces the oxidative stress and degrades photosynthesis pigments in plants [30].
Lead degrades the development of roots and arsenic effects yield of crop and chlorosis, plant height and decreases ability of seed for germination [44].
Nickel is important and considered as macronutrient in plants but present in excessive amount can inhibit root growth, short shoot yield, etc. [45].
Enzymes and co-enzymes both are made up various elements such as cobalt. High concentration of cobalt may cause depletion in nutrients like proteins, amino acids, carbohydrates, etc. Also exhibit retarded plant growth and development.
Photosynthesis is prime phenomena in plants and it requires iron element. The excessive concentration of iron can inhibit photosynthesis itself [24].
Plants experience oxidative stress upon exposure to heavy metals that leads to cellular damage and disrupt of cellular ionic homeostasis. To decrease the detrimental outcomes of heavy metal exposure and their absorption, plants have participated in detoxification processes highly based on chelation and sub-cellular compartmentalization. A primary class of heavy metal chelator known in plants is phytochelatins (PCs), are produced by non-translation from reduced glutathione (GSH) in a transpeptidation reaction catalyzed by the enzyme phytochelatin synthase (PCS) [39].
The various biosorption techniques adopted by the plants such as phytoextraction, phytoextraction, rhizofiltration, phytovolatilisation and many others.

Bioremediation of heavy metals by microbial strains
Various microbial strains can accumulate the toxicity of heavy metals from bacteria, fungi, algae and helps in bioremediation and biosorption [46]. Bacterial strains show five different mechanisms in resistance to heavy metals. These mechanisms are by inhibiting the entrance of metals into the cell. The cell wall, membrane and capsule prohibit entry of metal ions inside the cellular body. Carbonyl group in polysaccharides of bacterial capsule accumulates the ions of heavy metals. Ions of metal like zinc, lead, and copper resulted resistance by Pseudomonas aeruginous biofilm [47].
In bacteria, active transport illustrate largest group of heavy metal resistance. Active transport remove metal ions from cell membrane and it can be placed on either on plasmid or on chromosomes [48,49].
In intracellular sequestration, combination of metal ions to form large ion is done by several compounds inside cytoplasm of cell. Example; P. putida shows potential of intracellular sequestration of metal ions such as zinc, candium and copper [50].
In extracellular sequestration, metal ions are collected by periplasm or outer membrane of cells as insoluble compounds [51].
Condensation of metal ions was done by the bacterial strains. Strains decreasing chromate, vanadate and moyhybadate were observed from surroundings. Metal ions were utilized as electron donors for generating energy by bacterial isolates. Example: S. aureus strain for resistance of arsenic (As 5+ /As 3+ ) [52], Klebsiella pneumonia for resistance of mercury (Hg 2+ /Hg) [53].

Tolerance against heavy metals in bacteria
There are various processes of heavy metal resistance like extracellular barrier, extracellular sequestration, and active transport of metal ions (efflux), intracellular sequestration, and reduction of metal ions by microbial cells.
B. subtilis revealed the excessive potential to remove the amount of the cadmium.
Bacteria resistant to mercury are Alcaligenes faecalis, Bacillus pumilus, Pseudomonas aeruginosa, and Brevibacterium iodinium for the eradication of cadmium and lead metals. 59 isolated actinobacteria have shown resistance to the five heavy metals. Using molecular identification 16S rRNA, 27 strains were found to classified in the Streptomyces and Amycolatopsis genera [54].
Three strains were identified up to genus level based on their morphological, cultural, physiological and biochemical characteristics as Gemella sp., Micrococcus sp. and Hafnia sp. Among these three isolates, Gemella sp. and Micrococcus sp. exhibited the resistance towards lead, chromium and cadmium metals whereas Hafnia sp. exhibited reactivity to cadmium (Cd). All strains revealed dissimilar MICs against the heavy metals at different concentrations using Atomic Absorption Spectrophotometer [55].
Bacterial cell wall experiencing the metal ion is the primary constituent of biosorption. The metal ions get connected to the various functional groups such as (amine, carboxyl, hydroxyl, phosphate, sulfate, amines) exist on the cell wall of the microbe. The metal uptake mechanism involves binding of metal ions to reactive groups lies on cell wall followed by internalization of metal ions inside cell protoplast. Some metal in more amount are accumulated by Gram positive strains due to presence of glycoproteins in their cell wall. Fewer metal absorption by Gram negative strains is reported due to phospholipids and LPS in their cell wall.

Arsenic resistant bacteria
Gram positive and gram negative bacterial strains have been investigated in the absorption of heavy metals.
Arsenic resistant bacteria species are Enterobacter sp. and Klebsiella pneumoniae based on phylogenetic analysis of 16S rDNA sequence [56].
10 isolates of rhizobacteria out of which some were Gram-positive bacteria Arthrobacter globiformis, Bacillus megaterium, Bacillus cereus, B. pumilus, and Staphylococcus lentus), and few were Gram-negative bacteria (Enterobacter asburiae and Rhizobium radiobacter). R. radiobacter exhibited the highest MIC of greater than 1500 ppm of the arsenic metal [58].
Aeromonas, Exiguobacterium, Acinetobacter, Bacillus and Pseudomonas are isolates of bacteria that can tolerate high levels of arsenic species [59].

Cadmium resistant bacteria
Cadmium resistant bacterium, Salmonella enterica 43C is isolated from industrial effluent was characterized on the basis of biochemical and 16S rRNA ribotyping [62].
The efflux processes involves cadA and cadB gene method, and encodes several efflux pump proteins and various functional groups like amine, carboxyl, phosphate and hydroxyl ease cadmium binding sites to bacterial surface such as chemisorption. The membrane impermeablility is regulated by enzymes used in detoxifying the cadmium metal [63]. Various processes on the basis of morphological, biochemical characteristics, 16S rDNA gene sequencing and phylogeny analysis exhibited that the strain RZCd1 was recognized as Pseudomonas sp. M3. In log phase, industrial strains revealed more than 70% of the cadmium accumulation [57] (Table 2).

Mercury resistant bacteria
With the help of 16S rRNA gene sequence, Vibrio fluvialis CASKS5 strain was recognized. The mercury-absorption ability of V. fluvialis was examined at several amount of concentration and exhibit large MIC (Minimum Inhibitory Concentration) but low antibiotic resistance [68].
Highly mercury resistant bacteria strains were Brevundimonas sp. HgP1 and Brevundimonas sp. HgP2 with 16S rDNA from a gold mine situated in village Pongkor, West Java with high MIC of 575 ppm. The aim was to examine the effect of mercury on bacterial development and morphological changes of bacterial population. The development was observed by measuring optical density at 600 n [70].
Mercury-resistance in the bacteria isolates were classified into the various genera such as Pseudomonas, Enterobacteriaceae, Proteus, Xanthomonas, Alteromona, and Aeromonas [71].
Attachment to the cell membrane, influx and efflux adsorption, detoxification of toxic metals to less harmful form, the use of metallothionein protein were several processes for heavy metal resistance. Removal of the any ion can be decreased by efflux, an active extrution of the heavy-metal ion [72] (Table 3).

Lead resistant bacteria
Lead accumulation processes operated by the lead resistant bacteria isolates includes efflux mechanism, extracellular sequestration, biosorption, precipitation,  alteration in cell morphology, enhanced siderophore production and intracellular lead bioaccumulation [73]. Four distinct bacteria were isolated with high levels of resistance to lead, each exhibited resistance to 2 mM lead on the minimal medium. Two were identified as Gram-positive genus Corynebacterium and two were the Gram-negative genus Pseudomonas. Three strains transferred no observable plasmid, indicating that the metal resistance is encoded by chromosomal [74] (Table 4).
Lead-resistant bacteria play an important role in the development of leadexposed plants. The endophyte Bacillus sp. MN3-4 increases Pb(II) absorption in Alnusfirma, and Pseudomonas fluorescens G10 and Mycobacterium sp. G16 enhances plant development and growth and decreased Pb toxicity in Brassica napus [75].

Nickel resistant bacteria
The nickel-resistant bacteria were identified as Shigella, Enterococci and Enterobacter, but they were anaerobic, they only grew in the human samples from obese people and they tolerated a maximum concentration of 1 mM nickel [76].
AIcaligenes eutrophus H16 and N9A strains and derivatives of strain CH34 lacking one or another of its natural metal resistance plasmid were used as recipients. Both of the plasmid, pTOM8 and pTOM9 of strain 31A conveyed resistance features which were expressed except A. eutrophus H16 [79].

S. no.
Microorganism Accumulation of heavy metals in ppm References

Copper resistant bacteria
Copper-resistant bacteria have been isolated from the different sources, but copper-resistant Escherichia coli strains were isolated from agricultural sewage and phytopathogenic Pseudomonas and Xanthomonas strains.
The copA gene was noticed in the copper resistant strains Sphingomonas, Stenotrophomonas and Arthrobacter isolated from the contaminated soil from agricultural fields [81] (Table 6).
Bacterial strains showed high level of removal of heavy metals, determinants like YJ3 and YJ7 maybe resistance to Cu and isolates like SWJ11, MT16, GZC24 and YAH27 may be resistance to heavy metals such as Cu, Pb, Cd, Ni and Zn. It has been observed that plant growth-promoting bacteria can enhance the development and heavy metal uptake of plants [83,84].
Numerous bacterial species show resistance to heavy metal such as thallium, tungsten, uranium, plutonium, have been observed from sediment and water sample. Pseudomonas aeruginosa strains results in accumulation and resistance to these heavy metals. Plutonium is harmful for soil microorganism even at very low concentration and stops the growth of bacteria fungi present in soil and affects soil respiration [85].

Tolerance against heavy metals in fungi
Fungi are ubiquitous in nature and found in water and soil. Recent strains isolated from contaminated sites have shown exceptional potential to tolerate heavy metals [86].
Fungi show potential as biocatalysts to accumulate heavy metals and convert them into very less toxic metals. Fungi mostly use chelation method to upgrade the tolerance to harmful heavy metals.
Recent studies have concluded many fungal strains like Rhizopus stolonifer in tolerance to lead, cadmium, copper and zinc. Pleurotus ostreatus in strain is used in nickel resistance. Aspergillus niger lead to the removal of lead, zinc, iron by bioleaching process and Aspergillus niger lead to removal of Zinc, nickel, lead, cadmium, manganese by immobilized cells [87].
Fungus as biosorbents used in removal of heavy metal ions. Bioleaching involves use of heterotrophic fungi and their metabolic products for accumulation of heavy S. no.
In intercellular system, metal transport proteins show resistant by ejection of metal ions from inside the cytosol [88].
Fungi strains to tolerate heavy metals are Aspergillus foetidus and Penicillin simplicissimum.

Cadmium resistant fungi
Aspergillus versicolor, Aspergillus fumigatus, Microsporum species, Cladosporium species, Paecilomyces species, Terichoderma were investigated by results of Fazli et al. [89]. Biological mechanism of fungal isolate directly relies on resistance against cadmium metal. Paecilomyces species could accumulate 400 mg/L concentration of cadmium which is the highest MIC standard observed yet. Highly versatile fungus to cadmium stress was Aspergillus versicolor and most sensitive fungus species for inhibition of mycelia growth are Microsporum species and Cladosporium species. Unique and advance technologies in bio treatment of heavy metals are metal uptake technique natively, utilizing combination of isolates and cell structures manipulation by autoclaving [90] (Table 7).

Lead resistant fungi
Penicillin oxalicumis species acts as a biosorbent and removes lead from aqueous solution. The isolates reveals uptake ability and tolerance to lead are Aspergillus fumigatus, Penicllum simplicissimum etc. Fungus biomass which is physically and chemically retreated again was a technique applied for biosorption of lead metal [94] (

Mercury resistant fungi
Aspergillus niger and Aspergillus flavus used in bioremediation process in mercury contaminated soil. Both belongs to phylum Ascomycota and are soil fungi [95].
Fungal sensitivity against heavy metals alters the origination of fungal spores. Sporulation is a natural response created by fungi as metal avoidance strategy in heavy metal contaminated sites.

Nickel resistant fungi
Various fungi species such as Aspergillus niger, Aspergillus giganteus, Penicillin vermiculatum, Gliocladium species, Beauvaria species, Trichodermaviride and Rhizopusstolonifera induces shows sporulation due to increase in concentration of nickel in contaminated sites. Environmental factors like pH temperature organic matter and metal ions impacts toxicity of nickel. Alteration of magnesium transport minimizes nickel. Generation of chelating compounds like glutathione deactivates toxicity of nickel [97] (Table 10).
Microbes involved in biochemical mechanisms to exploit arsenic oxy-anions either as an electron acceptor (arsenate) for anaerobic respiration or as an electron donor (arsenite) to support chemoautotrophic fixation of carbon dioxide into cell carbon [102].

S. no.
Microorganism Accumulation of heavy metals in ppm References   Two arsenic resistant fungi are Fimetariella rabenhortii and Hormonema viticola were isolated from contaminated soil. In fungi, Evaluation of plant growth promoting factors. Arsenic shows resistance by mediation of phosphate solubilization. F. rabenhortii and H. viticola had capacity to produce indole acetic acid and siderophores [103].
acrA biosensor strain is first fungal biosensor for arsenic detection. Using fungi as whole cell biosensors have various advantages [104].
A non-pathogenic strain Aspergillus niger is broadly used in Industrial applications. Presence of lead and zinc does not affect the fungal spore growth ( Table 11).

Iron-resistant fungi
Iron is essential in low concentration but very harmful in high amount of concentration. The fungal strains useful in iron resistance are Aspergillus niger and Aspergillus foetidus and some Penicillium species too. Fungal strains have good ability for bio leaching process by interfering functional groups of enzymes [105] (Table 12).

Cobalt resistant fungi
Cobalt metal is found in state of cobaltite, linnaeite, smaltite, etc. Some fungal strains help in accumulation of cobalt are Aspergillus niger, Aspergillus foetidus and S. no. Microorganism Accumulation of heavy metals in ppm References

Tolerance against heavy metals in algae
Metal detoxification or chelation is one more strategy defense for heavy metal resistance. Algae secrete chelating molecules in response to metal ions that successively bind to them resulting in the sequestration of complexed metals in cellular organelles. Most of the algae strains are rumored to accumulate elevated metal ion concentration in cellular organelles. Additionally, the appliance of this metal resistance in biogenesis of metal nano-particles and metal compound nano-particles has been investigated by [107].
Algae are aquatic plants which absence of true roots and stems. Even when less nutrition is provided still they can grow in large biomass. Large size, high sorption ability and no production of harmful components are responsible for good biosorbent material. Features required for binding algae surface to heavy metal ions are algae species, ionic charge of metal and chemical composition of metal ion solution. Amine, carboxyl, sulfate, phosphate, sulfhydryl, hydroxyl, imidazole groups are metal ion binding sites on algal surfaces [108].
Algae show various mechanism such as formation of proteins which binds with metals, changes in structure of cell membrane, complexation or elimination of ions. Heavy metals can be eliminated for contaminated sites by either living cells or dead cells by usage of inactive biomass. Mechanism of absorption of living cells is very much complex than intracellular uptake [109].
Two processes in algal biosorption are involved. 1. Ion exchange method where ions present on algal membrane Ca, Mg, K, Na. They are displaced by metal ions. 2. Complexation between metal ions and functional groups. The metal removal process of algae is similar to bacteria by bonding of metal ions with the membrane [110].
Cladophora species are best bio indicator and scenedesmus species results in stress tolerance and accumulation of heavy metal like copper and chromium. In brown algae, cell wall contains fucoidin and olginic acid which helps in accumulation of heavy metals too [111].
Three fresh water microalgal determinants Phormidium ambiguum (Cyanobacterium), Pseudochlorococcum typicum and Scenedesmus quadricauda var. quadrispina (Chlorophyta) were tried for resistance and absorption of mercury (Hg 2+ ), lead (Pb 2+ ) and cadmium (Cd 2+ ) in aqueous solution. Transmission electron microscopy (TEM) was examined to contemplate the connection between heavy metal ions and P. typicum cells. At ultrastructural level, electron thick layers were recognized on the algal cell membranes when exposed to Cd, Hg and Pb [110] (Table 14).
Microalgae are also capable in utilizing the removal of heavy metals for water contaminated sites. Microalgae are unicellular organisms and also known as phytoplankton which are visible under microscope only and found in both fresh and marine water. Microalgae show positive responses in the resistance towards the heavy metals and convey better chances of bioremediation. Microalgae are also used as a bio-indicator to check or identify the effects of contaminants on ecosystem. Microalgae exhibit biosorption methods to accumulate heavy metals by showing extracellular mechanism and intracellular mechanism to deal with high toxic concentration. Microalgae mostly used to treat wastewater as it releases oxygen as a byproduct during process [123].
Bioremediation by Cynobacteria (Blue Green algae): Cynobacteria is efficient tool for enhancing the productivity of crop, and plants, formation of bio fuel, rise in fertility of soil and bioremediation also. To explore multiple functional bioagents, genetically engineered cynobacteria should be introduced heavy metals like cadmium, lead, copper, cobalt, manganese were treated with different cynobacterial species such as N. muscorum, A. subcylindrica, Nostoc, linckia, N. rivularis, etc present in sewage and industrial waste water [124,125].

S. no.
Metal Microrganism Biosorption of metals References
Cynobacteria acts as bioremediator because of their photoautotrophic nature and capability in nitrogen fixation. It is able to tertiary level of agro industrial effluents like oil refineries, paper and pharmaceutical industry. Nostoc species and Microcystis species accumulate wide range of organophosphate insecticides. As it is found in contaminated water sites and helps in high yield of plants and utilized for bioaccumulation. It can help to enhance the fertility of soil and useful as bio-fuels. It can be used as a good biofertilizers. Mechanism adopted by cynobacteria response to salanity result in bio-polymer production.
Cynobacteria develop bio-flocculants that shield there body mechanism from toxicity of heavy metas. Bio-flocculants are outlined by the presence of various negatively charged binding sides that permit cynobacteria in resistance of heavy metal from contaminated sites [126]. Cynobacteria have flourished numerous mechanisms for reducing the metal stress by intracellular metal sequestration, extracellular mechanism or binding of metals ions.
Metalithionein are metal binding proteins released by cynobacteria that support organism in metal sequestration of dangerous heavy metal ions.
Use of cynobacteria is much better than other microbes like bacteria fungi because of various other benefits like growth promoters, bio stabilizer, bio energy resource (bio-diesel), bio fertilizer, wasteland reclamation, carbon dioxide sequestration, methane oxidation.
Cynobacteria are very much efficient because of short generation time and helps in atmospheric nitrogen fixation.
Lichens in bioremediation: Lichens are made by symbiotic association of fungi and algae in which both benefit each other. In wastewater remediation, lichens used as a biosorbents.
In heavy metal contamination, lichens can be used as bio-monitors too and the capability to accumulate heavy metal allows the monitoring ability. Lichen Permelia perlata shows the potential in biodegradation in contaminated sites.
Lichens adopt numerous processes for metal uptake such as extracellular uptake by ion exchange method intracellular removal and capturing of metal particles. The studies done by UK researchers on lichen results that lichen reproduces on land contaminated with uranium particles from mining activities and lichen converts uranium into dark particles. Endolithic lichen can be studied as a future approach in field of bioremediation [127].

Conclusion
Heavy metal pollution are very harmful for humans, animals, aquatic species and plants too and they were accumulated on earth crust by natural process as well as human activities such as industrialization, urbanization, mining and extraction, agricultural practices, etc. Bioremediation is the process which use either naturally occurring or deliberately introduced microorganisms to consume and break down environmental pollutants, in order to clean a polluted site. Various studies had been done and various strains were investigated are above mentioned. Bacteria, Fungi, Algae all are helpful in maintaining tolerance against heavy metals in different contaminated sites. There are several microbes present that provide heavy metal resistance through develop different method of resistance against different heavy metal. It can reduce heavy metals from environment to some extent. Further research area needs to be extended on the focus of gene transfer within bio-films

Author details
Madhuri Girdhar*, Zeba Tabassum, Kopal Singh and Anand Mohan School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India *Address all correspondence to: madhurigirdhar007@gmail.com for Bioremediation and use of genetic modified organisms. These strategies would facilitate the development of improved techniques for the bioremediation of heavy metals in the environment.