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TREATMENT OF XENOBIOTICS OF INDUSTRIAL WASTE WATER BY MIXED CULTURED MICROBES

 

Clinical courses

About Authors:
Saptaparna Mukherjee
Emami Limited, Quality Control
West Bengal University of Technology
sapta.muk@gmail.com

Abstract
By polluted water (Industrial waste water) treatment we are trying to reduce the water pollution as well as the recycling of water may reduce the deficiency of water in dry places of earth. Xenobiotics are often synonyms of pollutants if this accumulates in the biots body and cause disease. Industrial waste water contents many pollutants like heavy metals, pesticides, toxic substances, which may affect the plant and animals of a particular geographic area and in taking by humans may cause severe diseases. Unlike to recent available filters our bio filter can remove the heavy metals (arsenic, cadmium, mercury, lead), the pesticides and any toxic substances found in industrial waste water (tannery, battery, paper etc) by microbial metabolism (Packed bed of fugue and bacteria) and other simple filtration(charcoal treatment, reverse osmosis etc). Microbes are very helpful in absorbing or chelating or degrading the heavy metals (arsenic, lead, mercury, aluminum, cadmium etc), pesticides, organic and radioactive substances. Like Aspergillus candidus (fugue), Rhodotorula rubra (yeast) and Thiobacillus ferooxidans, Serratia marinorubra (bacteria) can convert arsenic to asenite, which is less harmful than source. Similar microbes can find for other heavy metals and toxic substances.

This filter can be used as domestic and industrial purpose to reduce organic and inorganic load of industrial waste stream, the main importance is that the used bacterial and fungus beds are not pollutant for our environment. So the replacement of any unit of this filter will not cause any more pollution only reduce it. The waste unused water thus can be made to use as domestic and agricultural purpose. The main aims of our project are: waste water treatment, recycling and conserving water, save the animals and plants from xenobiotic effects due to industrial waste.

REFERENCE ID: PHARMATUTOR-ART-1948

1. Introduction
Projects of biotechnology are very helpful to lead a healthy life in this polluted earth. Nowadays pollution is a major problem of this green earth, every species of it is badly affected by water, air, land, sound pollution. By polluted water treatment we are trying to reduce the water pollution. Not only that but also the recycling of water may reduce the deficiency of water in dry places of earth.

2. Literature Review
Xenobiotics is a term which means chemical which is found in an organism but which is not normally produced or expected to be present in it. The term xenobiotic is derived from the Greek words
ξ?νος (xenos) = foreigner, stranger and βioς (bios, vios) = life. So these xenobiotics are often synonyms of pollutants if this accumulates in the biots body and cause disease. Industrial waste water contents many pollutants like heavy metals, pesticides, toxic substances, which may affect the plant and animals of a particular geographic area, and in taking by humans may cause severe diseases.


2.1 Effect of Arsenic on human health

  • Long-term exposure to arsenic in drinking water can cause cancer in the skin, lungs, bladder and kidney. It can also cause other skin changes such as thickening and pigmentation. The likelihood of effects is related to the level of exposure to arsenic and in areas where drinking water is heavily contaminated, these effects can be seen in many individuals in the population. Increased risks of lung and bladder cancer and skin changes have been reported in people ingesting arsenic in drinking water at concentrations of 50 |ug/litre, or even lower.
  • Long-term exposure to inorganic arsenic in drinking water in Taiwan has caused blackfoot disease, in which the blood vessels in the lower limbs are severely damaged, resulting eventually in progressive gangrene. Its occurrence in Taiwan may be influenced by factors such as poor nutrition. However, arsenic exposure has caused other forms of blood vessel disease in the limbs in several other countries.

2.2 Effect of Mercury on human health


  • Inorganic mercury is found in batteries and is used in the chemical industry and it is produced from elemental mercury through the process of oxidation. Inorganic mercury is the most common form that is present in drinking water but is not considered to be very harmful to human health, in terms of the levels found in drinking water. However, kidney damage may result from exposure to inorganic mercury through other sources.
  • Organic mercury (primarily methyl mercury) is produced by specific bacterial organisms in acidic surface waters that convert inorganic mercury into organic mercury, which is the form of mercury that poses a significant threat to human health. Methyl mercury is ingested typically by fish and bioaccumulates both in the tissues of fish and the humans that eat these fish.This form is rarely present in drinking water but is a very common contaminant in the tissues of fish and causes damage to the nervous system as well as teratogenesis.

2.3   Effect of Cadmium on human health

  • Cadmium waste streams from the industries mainly end up in soils. The causes of these waste streams are for instance zinc production, phosphate ore implication and bio industrial manure. Another important source of cadmium emission is the production of artificial phosphate fertilizers.
  • Cadmium is first transported to the liver through the blood. There, it is bond to proteins to form complexes that are transported to the kidneys. Cadmium accumulates in kidneys, where it damages filtering mechanisms. This causes the excretion of essential proteins and sugars from the body and further kidney damage. It takes a very long time before cadmium that has accumulated in kidneys is excreted from a human body.
  • Other health effects that can be caused by cadmium are Diarrhea, stomach pains and severe vomiting, bone fracture, reproductive failure and possibly even infertility, damage to the central nervous system, damage to the immune system, psychological disorders, possibly DNA damage or cancer development.

2.4   Effect of lead on human health

  • Lead and lead compounds are generally toxic pollutants. Lead (II) salts and organic lead compounds are most harmful ecotoxicologically.
  • Lead has been demonstrated to be toxic to a wide variety of organs in human. The organ system that have been shown to be most sensitive to low level exposure of lead.
  • Other effects include in nervous system (changes I neurotransmitter levels), metabolism (impairment of vitamin D metabolism), reproductive system (irregular estrus and decreased sexual hormone level), immune system (impaired lymphocyte function and impaired antibody formation).

2.5   Effect of pesticide on human body

  • The amounts and variety of pesticides now used are far greater than in any other time in history. The population is at great risk from the existing allowable levels of pesticide residues.
  • The data strongly suggest that exposure to these neurotoxic compounds at levels believed to be safe for adults could result in permanent loss of brain function when it occurs during prenatal and early childhood periods of brain development.
  • Pesticides may cause acute and delayed health effects in those who are exposed. Pesticide exposure can cause a variety of adverse health effects. These effects can range from simple irritation of the skin and eyes to more severe effects such as affecting the nervous system, mimicking hormones causing reproductive problems, and also causing cancer.

2.6 Effect of Toxic Substances on human body

  • The main toxic action includes the production of internal damage. Toxic effects produce liver damage. e.g. Carbon tetrachloride. Toxic effects produce kidney damage e.g. Some halogenated hydrocarbons.
  • The main toxic action is the depressant effect upon the Central Nervous System.
  • Cancer, asthma, chronic fatigue syndrome, allergies and multiple chemical sensitivities can be related to the unprecedented increase of toxic substances in our environment.
  • Microbes are very helpful in absorbing or chelating or degrading the heavy metals (arsenic, lead, mercury, aluminum, cadmium etc), pesticide, organic and radioactive substances. The important tolerance mechanism of xenobiotic by the microorganisms are (a)intracellular accumulation,(b)cell wall associated metal interaction,(c)extracellular immobilization of metals,(d)extracellular polymer metal interactions and (e)transformation and volatilization of metals.

Fig 2.1 : Various metal detoxification pathways in a microbial cell

We are using this technology to remove the pollutants of water to control water pollution, xenobiotic effect on human body, deficiency of water on dry earth.

Table-2.1 Microbes used in Waste Water Treatment

Removing substance

fungus

Bacteria

Heavy metal

Aspergillus candidus

Herminiimonas arsenicoxydans, Thermus thermophilus,Pseudomonas arsenitoxidans

1. Arsenic

2. Mercury

Aspergillus, Neurospora sp.

Clostridium, pseudomonas, E.coli, Bacillus megaterium, Aerobactor aerogenes

3. Cadmium

Rhizopus cohnii, Microsphaeropsis sp, Aspergillus niger,Candida glabrata

Bacillus circulans, lactic acid bacteria.

4. Lead

Aspergillus niger

E.coli, psudomonus, bacillous, some halophilic bacteria

Pesticides

Saprolegnia sp., Neurospora sp, mucor sp, aspergillus sp, penicillium notatum

E.coli, Corynebacterium sp, Erwinia sp., Micrococcus sp.

Toxic

substances(cyanide,

sulphides,phenols,surfact

ants,radioactive

substances,Ammonia,

chloramines,nonyl,

detergents)

Phanerochaete

chrysosporium,

P. ostreatus,

Saccharomyces

cerevisiae

Photobacteriumphosphoreum, E.coli

 

2.7 Brief description about the metabolism of different xenobiotic by different organism

2.7.1 Arsenic metabolism By Yeast:
Arsenic metabolism was studied for two marine microorganisms, an obligately aerobic yeast, Rhodotorula rubra. This was cultivated in media with (74As) arsenate (As V), and the products of arsenate metabolism were determined qualitatively. The yeast produced arsenite (AS III) and methylarsonic acid [CH3AsO(OH)2]. In additionhe yeast produced dimethylarsinic acid (CH3)2AsO(OH) and volatile alkylarsines. The yeast did not accumulate arsenite, but instead transported some of it into the culture medium and methylated the remainder first to methylarsonic acid and then to dimethylarsinic acid. Finally, the latter compound was methylated further and volatile alkylarsines were formed. Therefore the yeast convert relatively toxic arsenate, the most abundant arsenic compound in seawater, to products that were presumably less toxic.

By Bacteria:
Most of the arsenic in water is in the form of either As(V) compounds, called arsenates, or As(III) compounds, called arsenites. As(III) species are more toxic than As(V) species.The arsenic-oxidizing microorganisms oxidizes As(III) to As(V), which is then precipitated by the iron ions present in the water. As(III) exist as H3AsO3 which does not ionize enough to combine with metal ions, so As(III) is hard to remove by normal precipitation methods. Three clusters of genes involved in resistance to arsenic were identified. Quantitative analysis of the transporter-encoding gene mRNA demonstrated that the resistance operons are either constitutively expressed or induced in the presence of As[III] in H. arsenicoxydans. H. arsenicoxydans can accommodate a wide range of oxygen. Indeed, the H. arsenicoxydans genome harbors multiple respiratory pathways, permitting microorganisms to grow under aerobic, microaerobic, and anoxic conditions. Remarkably, the versatile regulatory system of H. arsenicoxydans enables it to sense dynamic changes in arsenic concentration and to initiate motility and EPS synthesis for attachment to this metalloid. Recent results suggest that microbial biofilms are involved in the adsorption and immobilization of metals (such as Pb[II] and Cr[III]).

2.7.2 Mercury Metabolism
Mercury (Hg) is one of the most hazardous contaminants and can be found in virtually all environmental media. Mercury may be present in different chemical forms in water systems,Hg(0), Hg(II), methylmercury (MeHg), dimethylmercury (Me2Hg), HgS and other Hg complexes. The chemical and physical properties differ among Hg species. This leads to a consequent change of its stability and mobility in different natural compartments and its toxic effects on living organisms. For example, organic Hg species are more toxic than inorganic Hg species. Due to its amphiphilie character, MeHg is highly mobile and may accumulate strongly in the food chain. Therefore, understanding the transformation processes of Hg in the environment is a key component of risk assessment of Hg in terrestrial and aquatic ecosystems and human health. Mercury in its elemental form (Hg(0)) is volatile and escapes to the atmosphere, thereby decreasing Hg concentrations in soils or waters. Microorganisms converting Hg into its volatile form are performing an important process of detoxification. So the mercury reducing microorganism such as Pseudomonas., entericbacteria, Staphylococcus aureus, Thiobacillus ferrooxidans, group B Streptococcus, Streptomyces, Flavobacterium, Achromobacter, Alcaligenes, Acinetobacter play an important role in mercury detoxification. Probable mechanism for mercury reduction:

The reduction pathway induced by the Mer operon.Mercuric ion is transported into the cell via a transporter protein encoded by MerT. Then, the organomercury lyase (gene product of MerB) catalyzes the protonolysis of C-Hg bonds in many alkyl and aryl Hg compounds producing a reduced organic moiety and Hg(II). The latter is then reduced by the mercuric reductase (encoded by MerA). The mercuric reductase transfers two electrons from NADPH via FAD to Hg(II). The result is a volatile loss of Hg(0). The two enzymes organomercury lyase and mercuric reductase interact and build a large hydrophobic pocket. For this reason these enzymescan have a broad-substrate specificity range (Lloyd, 2003; Barkay and Wagner- Dobler, 2005).

Cadmium is a highly toxic heavy metal element that is ubiquitous in the Earth's crust (ToxFAQs: Cadmium). Despite its toxicity, cadmium is widely used in industry. More than one half of the sites on the Environmental Protection Agency's National Priorities List are contaminated with cadmium. Microbial interactions with cadmium have been explored extensively in a wide variety of prokaryotes, fungi, and algae. Research has focused on cadmium transport and detoxification mechanisms, and cadmium immobilization by bioprecipitation and biosorption.

No essential biological function for cadmium has been identified, but a cadmium-dependent carbonic anhydrase has been reported in the marine diatom Thalassiosira weissflogii (Lane and Morel, 2000). Molecular mechanisms of cadmium uptake in microorganisms have not been well characterized, but uptake may occur via magnesium, manganese, or calcium transport systems (reviewed byNies, 1999). Plasmid -based cadmium efflux systems in bacteria have been identified. Gram-positive bacteria export cadmium via P-type ATPases (EC 3.6.3.3), and Gram-negative bacteria use Czc (cadmium, zinc, cobalt) divalent cation transporters for cadmium efflux (reviewed by Silver, 1998).

Cadmium-binding proteins have an important role in moderating cadmium toxicity in some fungi and bacteria. A metallothionein encoded by the CUP1 gene binds Cd2+ in Candida glabrata, the yeast cadmium factor (YCF1, EC 3.6.3.46) mediates accumulation of cadmium - glutathione complexes in Saccharomyces cerevisiae vacuoles, and metal -binding peptides ("phytochelatins") sequester cadmium in subcellular organelles in Schizosaccharomyces pombe and C. glabrata (reviewed by Perego and Howell, 1997). Cadmium-chelating proteins have also been discovered in filamentous fungi (Razak, 1989). Cadmium-binding metallothioneins have been identified in cyanobacteria (reviewed byTurner and Robinson, 1995), and cadmium-binding proteins have been reported in Pseudomonas putida (Higham et al, 1986) and Escherichia coli (Khazeli and Mitra, 1981).

The potential for microorganisms to immobilize or volatilize soluble cadmium has been explored. Direct reduction of cadmium has not been observed, but biomethylation of cadmium by polar marine bacteria has been reported (Pongratz and Heumann, 1999). Several microorganisms have been shown to precipitate soluble cadmium as insoluble sulfides (Holmes et al, 1997), phosphates (Montgomery et al, 1995), or carbonates (Cunningham and Lundie, 1993). Biomass from several bacterial, fungal, and algal species has been evaluated as biosorbents for the removal of soluble cadmium from solution (Volesky and Holan, 1995).

2.7.4 Lead Metabolism
(i) Intercellular Accumulation: This is a detoxification mechanism of Citrobactor by accumulating lead as PbHPO4.

(ii) Cell Wall Interaction: The immobilization of lead by exopolymers has been demonstrated in several bacterial strains including Staphylococcus aureus, Micrococcus luteus and Azotobactor spp.

(iii) Extracellular Precipitation: Metal resistant strains of Klebsiella aerogenes precipitate Pb as insoluble sulphide granules.

3. Practical Work

3.1 Materials, Chemicals and Instruments required

•       Five empty 1 liter bottle

•       One 5lt jar

•       Two stands

•       Pebbles

•       Sand

•       Saw dust

•       Activated charcoal

•       Two stopper

•       Pipe

•       Tap

•       Coconut thread

•       Sodium alginate

•       Calcium chloride

•       Czapecdox and nutrient media

•       Aspergillussp., E. coli.

•       A DC motor and agitator

•       Atomic Absorption Spectroscopy

3.2 Steps of the Project

3.2.1 Activated sludge process
This is a process for treating sewage and Industrial waste water using air and a biological flock composed of microorganisms. The microbes convert carbon into cell tissue and oxidized end products that include carbon dioxide and water. In addition, a limited number of microorganisms may exist in activated sludge that obtains energy by oxidizing ammonia nitrogen to nitrate nitrogen in the process known as nitrification.

3.2.2 Packed Bed of Fungus
A packed bed of fungus is used to remove pesticide, toxic substances from polluted waters of many industries as well as bound waters (pond, river etc).

3.2.3 Immobilized bacterial cell
A mixed culture of bacterial cell is immobilized by using sodium alginate and cacl2, and then the globules are placed in a bed of jute thread to remove heavy metals such as arsenic, mercury, cadmium, lead etc. from waste water of tannery, paper or battery factory.

3.2.4. Activated charcoal
When charcoal is activated, the surface area of each particle is dramatically increased. This increased surface area plays a key role in its adsorption ability, and activated carbon surface properties are both hydrophobic and oleophilic; that is, they “hate” water but “love” oil. When flow conditions are suitable, dissolved chemicals in water flowing over the carbon surface “stick” to the carbon in a thin film while the water passes on. Thus it removes the bad odor and color from polluted water.

3.2.5. Natural filtration
Three beds are used here for natural filtration of micro particles. Upper one is pebbles bed to remove larger particles, middle one is sand bed to remove medium particle and lower one is saw dust to remove fine particle.

3.3 Result and interpretation
To know the efficiency of this bio-filter samples (industrial waste water) were tested under Atomic Absorption Spectroscopy.

Atomic absorption spectrometry (AAS) is a spectroanalytical procedure for the qualitative and quantitative determination of chemical elements employing the absorption of optical radiation (light) by free atoms in the gaseous state.

3.3.1 Principle of AAS
The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on Beer-Lambert Law. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using Beer-Lambert Law.

Figure 3.3.1 Schematic diagram of AAS

3.3.2 Procedure and Results of testing by AAS
(i) First we have to prepare a no of solution of known concentration (1 ppm, 2ppm,3ppm,4ppm,5ppm and so on) of required ions to make the standard curve. Here we prepared solution of lead nitate, as Pb (II) is most common form of lead present in waste water.
(ii) The collected waste water was then run through the AAS.
(iii) After filtering the sample, this was again run through the AAS.
(iv) The absorbance is tabulated according to the concentration of lead and a standard curve is prepared.

Table-3.1 Summation of Observed data

 

Concentration(ppm)

Absorbance

Blank

0

0.00

Standard 1

1

0.17

Standard 2

2

0.34

Standard 3

3

0.48

Standard 4

4

0.65

Standard 5

5

0.83

Sample

?

0.75

Filtered Sample

?

0.06

3.3.3. Discussion and interpretation of the results
Now we can draw a standard curve taking concentration as x axis and absorbance as y axis, from this we can find the concentration of lead raw sample and Filtered sample.

Figure 3.3.2 Standard Curve of Pb

The green point is the concentration of lead in waste sample, which is 4.5ppm and the purple point is the concentration of lead in filtered stream which is 0.35, which is sufficiently less than the source.

3.3.4 Application of the Bio filter
* From the results it is clear that by this Bio filter any heavy metal; pesticide, toxic substances can be removed by choosing proper bacteria and fungi depending on the source of waste water.
* If a continuous flow of water is done this bio filter can be used up to 3 weeks.
* Handling and constructing of this bio filter is very easy and cheap.
* The water after purification by this filter is domestic useable.
* The output water is less harmful for life than the source of discharge.

3.3.5 Future Scope
* This bio filter can be used as medium scale water purifier in the industry where the waste water is discharged before it goes to the river or sea.
* The sludge produced after treatment can be used as manure after proper checking of microbial and chemical characters of sludge.
* Due to some obstacles we cannot verify other metals by AAS, but as per review of literature if proper microbes are selected, this bio filter will give satisfied result for all Xenobiotics.

4. Conclusion
Making something useful by own hand is really an achievement. We think this experience of making bio filter will help and encourage us for further projects. This project model may help other to make a real one by using proper microorganisms. The fungus are very sensitive so while making the bed one should be more careful in the preparation of culture medium(czapecdox) and in maintaining the exact pH(5.4), temperature(max 30º) etc. The sand, pebbles, charcoal should be clearly washed. Leakage of any part of the whole system should be clearly verified.

5. References
• Textbook of Environmental Microbiology by Pradipta K. Mohapatra,I.K.International, Chapter 13,Microbes in Metal Pollution Control.(2008)
• Environmental Pollution Control Engineering by C.S.Rao,New Age International Publishers, Chapter 9,Wastewater Treatment.(Second edition)
• Temporal Transcriptomic Response During Arsenic Stress in Herminiimonas arsenicoxydans, Jessica Cleiss-Arnold, Sandrine Koechler, Caroline Proux, Marie-Laure Fardeau, Marie-Agnes Dillies,Jean-Yves Coppee, Florence Arsene-Ploetze, Philippe N Bertin,2010.
• Arsenic Metabolism in Marine Bacteria and Yeast, F.V.Vidal and V.M.V Vidal,Scripps Institution Of 0ccanography,USA(1980)
• Metabolism and Toxicity of Arsenic: A Human Carcinogen,Pradosh Roy And Anupama Saha, Department of Microbiology,Bose Institute,Kolkata,India.(2002)
• Genes for all metals:a bacterial view of the periodic table. The 1996 Thom Award Lecture,Silver S,Department of Microbiology and Immunology, University of Illinois, Chicago 60612-7344, USA(1998)
• Microbial pathways for the mobilization of Mercury as Hg(0) in anoxicsubsurface environments,Heather Wiatrowski,Yanpin Wang, Pat Lu-Irving, Lily Young,and Tamar Barkay(2007)
• Lead mineral transformation by fungi,Sayer JA, Cotter-Howells JD, Watson C, Hillier S, Gadd GM,Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland, UK(1999)
• Biosorption of heavy metals by bacteria isolated from activated sludge.Leung WC, Chua H, Lo W, Department of Applied Biology, The Hong Kong Polytechnic University, Hung Hom, China(2001)

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