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Acrylamide with chemical formula C3H5NO is an organic compound. Synonyms for acrylamide are acrylamide monomer, acrylic amide, propenamide, 2- propenamide, acrilamida (DOT Spanish), acrylamide (DOT French), acrylamide solution , acrylic acid amide (50%), acrylic amide (50%), ethylene carboxamide, ethylenecarboxamide, propenamide (50%), propenoic acid, amide, RCRA Waste Number U007, UN 2074, and vinyl amide (Ware, 1989). Acrylamide is a chemical that commonly used in industry to prepare polymeric material that used in many products in contemporary living known as polyacrylamide. Acrylamide can be present in either monomer (single unit) or polymer (multiple units that joined together by chemical bonds) (The Merck Index, 1996). The polymeric form of acrylamide called polyacrylamide, known to be non toxic (Friedman et al., 2003), while the single unit substance is toxic and cause damage to the central nervous system, carcinogen in laboratory animals, producing an ascending central/peripheral axonopathy and also suspected to be carcinogen in humans.
2.1.1 Physical and chemical properties of Acrylamide
Acrylamide can occur both in crystalline solid and liquid form. It is colorless to white solid monomer, free flowing crystal that is soluble in water, dimethyl ether, ethanol, methanol, but is insoluble in heptane and benzene. The solubility of acrylamide in water at 30oC is 2155 g/L. The molecular weight of acrylamide is 71.08 Da, melting point of 84.5oC and 125oC boiling point at 25 mmHg (European Commission, 2002).
The crystalline acrylamide monomer is available as pellets of 98% and 95% purity. The specific gravity of acrylamide is 1.122 at 30° C. The 50% aqueous form is form for applications in which water can be tolerated. The monomer readily polymerizes at the melting point or under ultraviolet light (NICNAS, 2002). Solid acrylamide is stable at room temperature, but it may polymerize violently when melted or in contact with oxidizing agents. Commercial acrylamide monomer contains residual levels of acrylonitrile (1 to 100 mg/ kg) (IARC 1986). Table 1 shows the summary of physico-chemical properties of acrylamide while Figure 1 shows chemical structures of polyacrylamide and polyacrylamide.
2.1.2 Uses of Acrylamide and Polyacrylamide
Since the last century, the use of acrylamide has increased. Acrylamide is widely use in many industrial applications as well as for chemical and environmental purposes. It is known that the main use of acrylamide is in the production of high molecular weight polyacrylamide. Polyacrylamide is an important polymer to produce various compounds with different physical and chemical properties suited for industrial needed. It is estimated that almost 99.9% of acrylamide is used in the production of polyacrylamide in Europe Union (European Commission, 2002). In the paper manufacturing industry, polyacrylamide plays an important part as binders and retention supports for fibres and to retain pigments on paper fibres. In United Kingdom, approximately 12,000 tons of polyacrylamide is used in the paper production industry annually.
The physical properties of polyacrylamides are decided by copolymerizing with a variety of different vinylic monomers. Polyacrylamide can be manufactured as cationic, non-ionic, or anionic polymer. Both cationic and anionic polyacrylamides are produced by the process of copolymerisation of acrylamide. Cationic polyacrylamides are useful for flocculation of sewage sludge and various industrial wastes, as well as retention aids in the paper industry (Barvenik, 1994).
Large quantities of acrylamide are used in the production of polyacrylamide gel as a grouting agent to stabilize mineshafts in the mining industry, tunnels, and dams to increase the strength and to restrict the flow of water through a structure (Mona et al., 2001). In the mining process, polyacrylamides are used as flocculants to separate solids from aqueous solutions. It is also used in the disposal of industrial wastes and in the cleansing of water supplies (European Commission, 2002). Polyacrylamide allow more concentrated sludge when they are used as sludge conditioning or dewatering agent than inorganic coagulants. When polyacrylamide is used in potable water treatment, it must not exceed of 0.05% of its monomer; however there are polyacrylamides containing 0.1-5% of monomer used as industrial coagulants (Croll et al., 1974). The principle of the coagulation process is when the polymers bind with the particles, it will form heavy aggregates that quickly settle out of solution and leave clear supernatant (Barvenik, 1994). The most effectual polymer is the one that have high molecular weight cationic polyacrylamide (1.5 X106 g mol-1) as it obtains a high exclusion efficiency % with dosage as low as 64 mg L-1 (Arifin et al., 2004).
Acrylamide also used to decrease soil erosion, and this part has received rising of attention in recent years. The most extensively published is in furrow irrigation systems, in which polyacrylamides is added to the irrigation water to evade erosion of the furrows (Lentsz et al., 1992). Polyacrylamide adds to the irrigation water will reduced up to 94% of furrow erosion. Polyacrylamide has been shown to reduce erosion when introduced through a sprinkler irrigation system (Byornberg and Aase, 2000; Green et al., 2000).
Smaller quantities of acrylamides are formulated in cosmetics and soap preparations as thickeners. It is also use in dental fixtures, preshave lotions, and hair grooming preparations. This compound also use as permanent press fabrics, in molecular biology applications, photographic emulsions, and food processing industry; in the production of diazo compounds; and for gel chromatography and electrophoresis (Sittig, 1985; IARC, 1986). In the textile industry, acrylamide polymer, polyacrylamides are used to size and shrink-proof material and as water repellents. Home appliances, building materials, and automotive parts are coated with acrylamide resins and thermosetting acrylics.
Polyacrylamide gel is used as a medium for hydroponically grown crops, and as a binder of bone cements (European Commission, 2002). Hydroponics is a conventional method in horticulture technique. This technique allows crops to grow faster and more consistent in quality than conventionally grown produce. This is because; polyacrylamide that used in this technique is essentially inert and has high ability to absorb water to supply for the crops and capable of holding moisture.
There is no specific data regarding the usage of acrylamide and polyacrylamide in Malaysia; however, several industries in Malaysia use polyacrylamide. The industries that use the most polyacrylamide are in waste water treatment, paper, and pulp processing. For the golf course in Malaysia, polyacrylamide was used to toughen the foundation of the artificial lakes. These cause the contamination of underground water supplies and therefore cause several poisoning and disorders of the central nervous system. Chatterjee (1993) has reported that many golfer, caddies, as well as local peoples have been found to endure irritations, skin diseases and other allergic symptoms.
In agricultural area, polyacrylamide is used as a stabilizer (25-30% solutions) in herbicide glyphosate (ROUNDUP™) formulation. Polyacrylamides are mixed up with various organic solvents forming thickening agents, and then combined with herbicides to increase the herbicides surfactants capabilities (Bouse et al., 1996). According to Mansor (1996), the glyphosate is the most famous herbicide used to solve the weeds problem all over Malaysia. This herbicide is used for the control of a wide range of broad-leaved weeds and grasses in agricultural estate crops such as rubber, oil palm and cocoa. It is estimated up to 8 million litres of glyphosate were used in year 2000 and from that data, at least 2 million litres of polyacrylamide is dumped into the soil and the rivers every years (AGRIQUEST, 2000).
Generally, most part of polyacrylamides is nontoxic. However, due to the polymerization process, these polymers can have a residual of its monomer, acrylamide; a peripheral nerve toxin (McMollister et al., 1965). The range of acrylamide that might contaminate polyacrylamide is in between 0.05 to 5.0% of the final product (Croll et al., 1974). After flocculation with polyacrylamides, acrylamide will remnants in the water due to its high water solubility and have high tendency not to be absorbed by sediment and sludge, although some of it may be trapped in the sediment (Brown et al., 1980).
2.1.3 Production of Acrylamide
For the production of many chemical compounds, usually large amount of energy needed to generate the reaction, and this can adversely affect the environment. Even though there are many alternative biotechnological production processes exist, they are often hampered by economics, although it is known to have potentially beneficial with respect to environmental protection. Nevertheless, increasingly severe environmental constraints will favor processes that can be done under milder conditions (Yamada and Kobayashi, 1996).
Usually biotransformations are used commercially when conventional chemical approaches are too expensive. In contrast to the conventional usage of fine chemicals, commodity chemicals used in biotransformation are low-priced, have a larger demand, and are produced and sold in high volume. In addition, commodity chemicals are characterized by the low cost of raw materials compared with the cost of production which characterizes fine chemicals. One of the most important commodity chemicals used in coagulators, soil conditioners and stock additives for paper treatment and paper sizing, and for adhesives, paints and petroleum recovering agents is acrylamide.
Conventional chemical synthesis involves hydration of acrylonitrile with the use of copper salts as a catalyst. However, this chemical method has various problems: (i) the rate of acrylic acid formation is higher than that acrylamide formation, (ii) the double bond of both substrate and product causes the formation of by- product such as nitrylotrispropionamide and ethylene cyanohydrate and (iii) polymerization occurs at the double bond of both substrate and product. Figure 2 shows the comparative flow sheet for the microbial and conventional processes.
For that reason, the enzymatic conversion from acrylonitrile to acrylamide could be done by microorganisms' catabolyzing acrylonitrile. Since it is inhibitory for nitrile hydratase activity when added to the reaction mixture at a high concentration, acrylonitrile, which functions as a substrate, was added in small portions to the mixture. More than 99% of the substrate (in this case, acrylonitrile) was converted to acrylamide without formation of any by products (Yamada and Kobayashi, 1996).
2.2 Acrylamide as pollutant
The contamination of acrylamide monomers in the environment through the use of polyacrylamide in china clay, paper industry, and water industry have occur long ago (Bachmann et al., 1992). The exposed workers will have symptoms of numbness, limb pain, peeling skin, and sweating hand. Acrylamide monomers can enter the environment by many ways. In the acrylamide production, closed system is now being used. Thus, the production processes of acrylamide are unlikely to be source of environmental contamination, except if there is a problem such as leaking from the reactor. Acrylamide discharge to water and environment also occur from acrylamide-based sewer grouting and wastepaper recycling.
Because of the high water solubility, acrylamide can easily contaminate water system and not likely to be source of air contaminant due to its low vapour pressure. The usage of polyacrylamide flocculants that contain residual levels of acrylamide monomer is a major source of drinking water contaminate by acrylamide. Public can expose to the acrylamide contamination from the polyacrylamide that used as flocculants agent in water treatment system (Brown et al., 1980, Howard, 1989). In potable water treatment, polyacrylamide containing in it must not exceed more than 0.05% (w/v) monomer. It may not be removed in most of the technique used in water treatment process (Croll et al., 1974). Igisu et al., (1975) has reported the cases of acrylamide poisoning in Japan as a result of acrylamide contaminate the water supply. Acrylamide was found to remain in tap water at least for two month after flocculation with polyacrylamides as it is water soluble and is not readily absorbed by sediment (Brown et al., 1980).
The recommended standard for drinking water in Malaysia was set on the basis of WHO Guidelines of 1976. The maximum allowable level of acrylamide containing in drinking water of Malaysia is 0.0005 mg/L (National Technical Committee, 2000). In both tap water and river area where polyacrylamide ware used for the treatment of potable tap water, acrylamide was detected at levels of less than 5µg/L. In West Virginia (USA), acrylamide was reported to contain 0.024 - 0.041 mg/L in the sample collected from public drinking water supply (Brown and Rhead, 1979).
In Malaysia, report by The Environmental Quality Report (1997) showed that a total of 908 water quality stations along 117 rivers were monitored by six parameters namely pH, dissolved oxygen, biological oxygen demand, ammoniacal nitrogen and suspended solids are taken for consideration to compute the quality index. Based on the calculation by 1997 WQI, only 24 rivers were categorized as clean, 68 slightly polluted and 25 rivers polluted. In terms of NH3-N (Ammoniacal Nitrogen) contain in the river, a total of 93 rivers were categorized as polluted. This is largely due to livestock farming and domestic waste (Malaysia Environmental Quality Report, 1997).
Environmental pollution of acrylamide may caused by the disposal or leaching of residual monomer from polyacrylamides. Report from the Toxic Chemical Release inventory (TRI) shows that estimated 5, 912, 663 lbs were released to the environment from 43 facilities that manufactured or used acrylamide in United States in 1996 (TRI96, 1998). In 1999, the number of facilities increased to 90, and the amount of acrylamide released is drastically increased up to 7, 542, 385 lbs. 99.6% of the total acrylamide released was on the underground soil injection. Statistic shows that 24, 874 lbs of acrylamide were released to air from forty-four facilities. Twenty-one facilities, each were found to release >100 lbs of acrylamide to air, which represented 98.7% of the total emission (TRI99, 2001). Other 1999 from the total released with the amount of 370 lbs were to water and another 6, 289 lbs to land (TRI99, 2001).
Acrylamide also can be exposed to the working environment. Even the small amount of polyacrylamide used in industry can resulted in acrylamide contamination in the workplace. Exposure of acrylamide can occur during the production of acrylamide and polyacrylamide, during acrylamide grouting, and also during chemicals preparation in laboratory. People who work in industries that use acrylamide and polyacrylamide such as in construction, plastics manufacturing, cosmetics, paper and pulp, mining, and agricultural industry are potentially exposed to acrylamide poisoning. There is no report regarding acrylamide exposure and poisoning for grouters, but the exposure for these particular person may be greater than workers in other industry (WHO, 1985).
1950s is the year where acrylamide grouts were started to use in USA (Mona et al., 2001). Acrylamide grouts generally consist of a 19:1 mixture of acrylamide and a cross-linking agent (EU Risk Assessment Report, 2002). In the end of 1970, the production of acrylamide grout in USA dropped because of the human health concern by the operators. However, in 1989 approximately 43% of grouts still be used in USA (Mona et al., 2001).
In 1997, product Rocha-Gil (Siprogel) that contains acrylamide and methylolacrylamide (N-hydroxymethylacrylamide) was used in the construction site for building tunnel in Hallands?sen, Sweden and in Romeriksporten, Norway (Mona et al., 2001). In both areas, the water released from the construction sites causes the high concentration of acrylamide in recipient's waters (Swedish Environmental Protection Agency, 1997); Sverdrup et al., 1999; Kallqvist et al., 1997).
In the middle of the various uses of polyacrylamide, it is mixed with variety of organic solvents to form thickening agents that are then combined with herbicides (i.e., glyphosate) to boost its surfactant capabilities (Bouse et al., 1986). For the commercial herbicide, polyacrylamide is used as additive (25% to 30% solutions) to reduce spray drift and to act as a surfactant (Smith et al., 1996). Glyphosate formulation can be more toxic than glyphosate alone, for example: RoundupTM can be 30 times more poisonous to fish than the glyphosate itself (Servizi et al., 1987).
Studies show that heat (Burrows et al., 1981), light (Reddy et al., 1994), and environmental condition (Kay-Shoemaker et al., 1998), promoted the depolymerisation of polyacrylamide to acrylamide. Photodegradation of polyacrylamide is a major factor in environmental degradation.
There are no reports available regarding acrylamide concentration contaminate in plants and food products. Suggested that plants and foods product may expose to acrylamide via air or contaminated water during growth or manufacture. However, acrylamide may present in foods result from the Maillard reaction between the amino acid asparagine and certain reducing carbohydrates when the foods is prepare at high temperatures (Mottram et al., 2002; Stadler et al., 2002). Acrylamide level will increase with the time of heating. Maillard reaction is a reaction that produces the tasty crust and golden colour in fried and baked foods (Friedman, 2003).
In heated protein-rich foods, moderate levels of acrylamide (5-50 mg/kg) were detected and even higher content were detected (150-4000 mg/kg) in carbohydrate-rich foods, such as potato, beetroot, also commercially potato products and crisp bread. Acrylamide cannot be detected in unheated or boiled foods (<5 mg/kg).
2.3 Toxicity of Acrylamide
Yang et al. (2005) has evaluated the toxicity of acrylamide. The result showed the mutagenic potency of acrylamide for Salmonella strains TA98 and TA100. Mice that exposed to acrylamide via intraperitoneal injection at dose of = 50 mg/kg shows an increasing in the incidence of chromosomal aberrations in its bone marrow cells (Chiak and Vontorkova 1988). Marlowe et al., (1986) study shows that group of mice received single of oral dose of 116-121 mg/kg. Acrylamide has been found in epithelia of oral cavity and oesophagus, liver and gall bladder. High concentration of acrylamide was present in kidneys, testis, and pancreas. Besides causes several histopathological lesions in the seminiferous tubules, acrylamide also shows effects of toxicological on male rat's reproductive system. Research by Ikeda et al., (1987) suggested that persistence of acrylamide or its metabolites in red blood cell following repeatedly exposure of acrylamide in dogs and pigs.
Acrylamide can damage nervous system, causing numbness and weakness in the hand and feet. Acrylamide can affect human health through inhalation, absorption through skin, causing a rash or burning feeling on contact. It can also cause loss of balance, slurred speech, and heavy sweating. Contact may cause eye burns and a skin rash. Approximately 20, 000 workers were potentially exposed to acrylamide in 1976 (NIOSH, 1984, IARC 1986).
Acrylamide is a toxic three-carbon compound containing an amide group and an a,ß-unsaturated olefin bond (David et al., 2005). This compound will reacts with nucleophilic compounds via a Michael addition. It exerts toxic effects by forming adducts to nucleophilic moieties such as sulfhydryl groups containing proteins and amino acids (Barber et al., 2001). Human exposure to acrylamide is primarily occupational from dermal contact. Acrylamide or its metabolites bind to RNA, DNA, and protein in a range of tissues.
There is a study in China on workers who were exposed to acrylamide by inhalation between one month and 11.5 years in an acrylamide synthesis room. Blood samples were obtained from 41 workers on that industry (Bergmark et al., 1993) and hemoglobin (Hb) was extracted for analysis of acrylamide and glycinamide adducts. Workers were potentially exposed to acrylamide by inhalation and dermal exposure. Skin peeling was observed on the hands indicate that the dermal exposure occurred. In report by Donovan and Pearson (1987) severe symptom of acrylamide toxicity detected within 3 hours of deliberate oral ingestion of acrylamide. This indicates that rapid and extensive absorption of acrylamide by the oral route. Hagmar et al., (2001) report his study on the effects of very high exposure of acrylamide on workers works in Chinese factory manufacturing acrylamide or using acrylamide or using acrylamide in China. 210 workers were found to expose to grouts containing acrylamide for approximately 2 months, and there was significant connection between hemoglobin adduct levels and exposure categories.
Many studies have been conducted to assess neurotoxic effects of acrylamide in occupationally-exposed workers (Deng et al., 1993). People who have been exposed to acrylamide poisoning will experience early symptoms like skin peeling from hands; weaken legs, numbness hands and feet, and impairment of the vibration sensation in the toes and loss of ankle reflexes. In Japan, the contaminated water supply with acrylamide cause several cases of acute acrylamide poisoning. Igisu et al. (1975) reported a level of 400 mg acrylamide/L in well-water in Japan that had been contaminated from a grouting operation 2.5 metres away. Through ingestion and external use of the well-water that readily contaminated with acrylamide, reportedly that there were five people that have been exposed and experienced the acrylamide toxicity. Intoxication symptoms included memory disturbances, confusion, hallucinations, disorientation and truncal ataxia. But within four months period of time, all of the exposed persons have completely recovered (Igisu et al., 1975).
Beside human, acrylamide also believe to cause abnormalities in mitotic and meiotic in both animals and plants (Shairashi, 1978; Shanker et al., 1987). The metabolization of acrylamide to epoxide, glycidamide (2,3- epoxypropanamide), shown to have neurotoxic potential (Barber et al., 2001). High concentration of acrylamide in drinking water of rats resulted in increased of tumor production at the tunica vaginalis testis (Damjanov and Friedman, 1998), adenomas of follicular cells of the thyroid (Johnson et al., 1986, Friedman et al., 1995), and tumor of the mammary fibroadenomas (Crump, 1999). The mode of action appears to involve binding of acrylamide to dopamine receptors (Crump, 1999a; Crump, 1999b).
Many early reports suggested that acrylamide toxicity was mediated by multifocal swellings and degeneration of long myelinated axons in the central and peripheral nervous systems (Gold and Schaumburg, (2000); LoPachin and Lehning, (1994); Spencer and Schaumburg, (1976); Spencer and Schaumburg, (1977); Tilson, (1981). Recent studies have been conducted in rats and demonstrate dissociation between development of distal axonopathy in the peripheral nervous systems and classic neurological signs such as ataxia, hindfoot splay and hindlimb muscle weakness. These symptoms observed in rats that exposed to acrylamide dosage ranging of 20 mg/kg or 50 mg/kg per day. But the development of anoxypathy observed only when the rats is exposed to a low dosage of acrylamide, in long term condition (21 mg/kg per day) (Lehning et al., (1998); Crofton et al., (1996). This lack of correspondence proposed that axon degeneration did not play a major role in the development of acrylamide neurotoxicity. Therefore, the hypothesized that axonopathy is a non- specific effect of acrylamide in fact it is related to duration of acrylamide exposure (LoPachin et al., 2000).
Acrylamide grouts that used in the building of tunnels in Hallandsasen, Sweden, and in Romeriksporten, Norway in 1997. Large-scale of Rhoca-Gil used to build the 8.6km tunnel in Hallandsasen (Mona et al., 2001). After a few week of injection, the symptoms of acrylamide poisoning were observed in fish and cattle downstream the site of the project. Not only that, the symptoms of acrylamide poisoning also were observed in people who works in the tunnel (Swedish Environmental Protection Agency, 1997). Project in Romeriksporten is to build a 14km long railroad tunnel. The construction site is located in the middle of Olso City, main airport Gardermoen. The large water leakage makes the company decided to use Rhoca-Gil 110/25 as grouting agent to stop leakage of the water (Mona et al., 2001). 73 workers were examined by the Norwegian Occupational Health Services. From that number, 4 workers detected to have skin effects due to acrylamide exposure. Another 7 workers were found to have slightly reduction of nerve (Kjuus et al., 1998). Based on the incident that occurred, Norwegian government come to a decision to ban the use of acrylamide- and methylolacrylamide used as grouting agent for the construction site in the late 1997 (Norwegian State Pollution Authorities, 1997).
Manufacturing, processing, and handling large-scale and large quantities of chemicals have led to serious surface, subsurface of soil and water contamination with variety of dangerous and hazardous toxic compounds. There was a large increase in the diversity of organic compounds that are industrially produced and which were carelessly release into the environment. Chemicals which have been synthesized in large quantities differ substantially in chemical structure from natural organic compounds. The chemicals are designated as xenobiotic because of their relative recalcitrance to biodegradation (Singh and Ward, 2004). The accumulation of many chemical compounds in the environment, particularly in soil and water are significant concern because of their toxicity, as well as their carcinogenicity properties and also because of the high potential of the compounds to accumulate in living systems.
Biodegradation is an important removal mechanism for the large quantities of chemical that released in our environment. The result of biodegradation is a decrease in the quantity of chemicals present in the environment and is a vital technique in preventing the accumulation and persistence of the chemicals (Shimp et al., 1990). Biodegradation of organic chemicals is one of the critical processes that determine the fate and behaviour of xenobiotics in the environment.
The ability of microbes to degrade pollutants into harmless constituents has been explored as a mean to biologically treat contaminated environments, and today it is the subjects of many research investigations and real-world applications (Jim et al., 2005). Now, it is also the being the basis for the emergent field of bioremediation.
Bioremediation can be defined as any process that uses microorganisms or their enzymes to return the environment altered by contaminants to its original condition. Mueller et al., (1992) defined the bioremediation is the use of microorganisms, plants or biologically active agents in order to degrade sequester and conjugate environmental pollutants.
Attention in bioremediation for the polluted soil and water has increased in the last two decades mainly because of the ability of the microorganisms to degrade the toxic compounds which were known to be resistant to the natural biological processes in the environment. It is known that the microorganisms in the environment oxidize many natural products and man-made compounds to carbon dioxide (CO2) and this constitutes an important part of the carbon cycle on the earth (Dagley, 1975). These microbes possess various enzymes capable of converting the toxic environmental pollutants into non-toxic compounds, which may serve as C and N substrates (Ingvorsen et al., 1991).
The uses of microorganism for bioremediation process determine by the condition of the contamination site whether it can be used indigenous or exigenous. Generally, many of the degrading-microorganisms used for this process are naturally occurring and members of stable microbial communities (Lindstrom et al., (1991); Stevens et al., (1991). Nowadays, in order to enhance the degradation capabilities of the microorganisms, recent research has developed the use of genetic engineering and selection techniques for the isolation of the target microorganisms.
2.5 Degradation of polyacrylamide to acrylamide
As discuss earlier in this part, acrylamide polymer, polyacrylamide is one of the major compound in industrial application. Polyacrylamide is used as flocculants agent in water treatment process to increase the process of sludge thickening and dewatering. It is also used as cement binder (Dos Santos et al., 2003), in sugar refining (Bologna et al., 1999), as well as formulation with pesticides to increase the surfactant capabilities.
Studies by Marcus et al., (2002) showed that temperature, light, biological, chemical, as well as mechanical promoted the degradation of polyacrylamide to acrylamide. Result from study by Eldon, (1997) indicate that photolytic effects or energy cause polyacrylamide degradation to acrylamide. The light energy can break chemical bonds, thus, degrading polymers (Decker, 1989; Rabek, 1987; Grassie and Scott, 1985). It is in line with report by Crosby, (1976) suggested that polyacrylamide is compose of C-C, C-H, and C-N bonds. Those bond strengths are approximately 340, 420, and 414 kj/mol, respectively, and these bonds can be broken down by wavelength of 325, 288, and 288nm, respectively.
The degradation of polyacrylamide by microorganisms as source of nitrogen for the microorganism's growth has been examined by Kay-Shoemake et al., (1998). Result obtained indicates that there were present of enzymes capable of biological hydrolysis of the amide to form NH3 and an acid. However, the study of polyacrylamide degradation for carbon source of the microorganism growth showed negative result. This can conclude that the bacterium studied not containing required enzymes to degrade the carbon backbone of polyacrylamide to satisfy their carbon requirement.
2.6 Degradation of acrylamide
Polyacrylamides for the most part are non-toxic. However, after polymerization, these polymers can have a residue of acrylamide, a known peripheral nerve toxin (Eldon et al., 1997). On release to the environment, acrylamide may undergo a number of degradation depending upon the place into which the release occurs. Due to its high water solubility, most probably acrylamide will be eliminated from the atmosphere by rain out (European Commission, 2002).
The degradation of acrylamide in river was studied by Brown et al., (1980). Two parameters were used in this study, which were sterilized river water and non-sterilized river water. Tested river water was added with acrylamide monomer at concentration of 0.5 and 5 mg/L. Then, the samples were stored under anaerobic conditions. No degradation was observed in any samples after 41 days incubation. For non-sterilized river water, degradation was observed. The absence of acrylamide degradation in sterilized river water proposes that the degradation occurs through a biotic process, with abiotic process such as hydrolysis and photolysis being negligible.
There is a report regarding the considerable acclimation period required for the removal of acrylamide in water (Croll et al., 1974; Brown et al., 1980) compared to soil where acrylamide monomer was rapidly hydrolyzed (Abdelmagid and Tabatabai, 1982). Acclimated microorganisms completely degrade acrylamide in 2 days, compare to non-acclimated microorganisms where 12 days needed to completely degrade the same concentration of acrylamide at 10-20 ppm in river water (U.S EPA, 1985).
Many species of microorganisms has ability in degrading acrylamide as their carbon or/and nitrogen source for growth. These tiny organisms can degrade acrylamide under light or dark, as well as in anaerobic or aerobic conditions (Brown et. al., 1980). Wang and Lee (2007) have reported regarding the ability and performance of bacteria on degrading acrylamide using Ralstonia Eutropha. The result indicated that the bacteria could remove acrylamide at concentration below 1446 mg/L. There is also a report regarding acrylamide degradation by bacteria under aerobic and anaerobic condition. Under aerobic condition, Pseudomonas stutzeri could utilize acrylamide at maximum concentration of 440 mg/L (Wang and Lee (2001). The acrylic acid and ammonia by-products were used as carbon and nitrogen source, respectively.
2.7 Effects of Heavy metals
Heavy metals contamination has been one of the environmental problems nowadays. The spread of heavy metals in the environment can be caused by the waste products disposal (Lasat, 2002). Beside that, it is also due to such industries that involve in metal processing, combustion of wood, coal and mineral oil, and from the use for plant protection (Schinner and Klauser, 2005). The widespread of heavy metals in environment not only affected soil, water resources and endanger human health (Shi et. al., 2001), but also results in the reduction of microbial diversity and activities in soil (Lasat, 2002; Akmal et. al., 2005). Soil microorganisms play an important part in recycling the mineral and in the decomposition of organic matter (Wardle and Ghani, 1995). It is also important in the degradation of others toxic compound in the soil. The used of microorganisms for the environmental bioremediation purposes can be disrupted by the present of heavy metals in the bioremediation site. So, this is one of major limitation aspect in bioremediation.
The presence of heavy metals in environment decreased the activity and diversity of microorganisms in the environment, and disturbing the balance of population interactions within the community (Wang et. al., 2010). Most of the previous studies has been conducted involving soil microorganisms, stated that toxicity of heavy metals has been related to their total concentration in soil (Huang and Shindob, 2000). The higher the heavy metals concentration, the greater their effect on microbial community. In addition, report by Zarnovsky et. al., (1994) shows that activated sludge microorganisms and process efficiency were inhibited by cadmium, chromium, and nickel at concentrations above 10 mg/L. Heavy metal exert their toxic effect by interaction with enzymes, thus cause inhibition of many metabolic processes within the microorganisms (Gassic and Korban, 2006).
2.8 Cell immobilization
Immobilization of microbial cells is a new approach in biotechnology especially in the bioremediation area to remove the environmental contamination. Immobilization of microbial cells is a process which involve the entrapment of living microorganism's cell by using a semi-permeable polymeric gel structure (Moslemy et. al., 2002). The technology of cell immobilization has been reported to have been successfully used in bioremediation purpose (Cassidy et. al., 1996).
Studies by previous researchers reported that there are many advantages of using immobilized cells for the removal of environmental pollutants over free-cells formulation. The used of gel matrix in cell immobilization technique can protect the microbial cells from biotic stress (Smith et. al., 1996), and also from abiotic stress such as the toxic compounds inhibitory effect (Cassidy et. al., 1997). Beside that, it also enhanced the microbial survival as well as improves the physiological activity (Weir et. al., 1995). Cell immobilization also has high productivity, as the cells are confined and the cell densities are high (Lee et. al., 1994).
There are many methods proposed to immobilize microbial cells. These methods include cross-linking, cell entrapment, and encapsulation. In cell immobilization technique, one important criterion is to use immobilization matrices with high integrity to ensure that it can prevent the microbial cells from leaking out into the environment (Premkumar et. al., 2002). Ideal matrix for immobilization method should have the ability to prevent cell flow within the matrix, functional at extreme temperatures, can be used in inconsiderate water conditions for example contaminated water, and allow the flow of oxygen and other nutrients through the matrix. Commonly used immobilization matrices include alginate beads, polyacrylamide gel, activated carbon, agarose, k-carrageenan, and gellan gum beads (Chung et. al., 2003; Somerville et. al., 1977; Bandyopadhyay et. al., 1999; Knaebel et. al., 1996; Audet and Lacroix, 1989; Moslemy et. al., 2003).
Several type of yeast and bacteria strain such as Pseudomonas aeruginosa (Prabu and Thatheyus, 2007), Acinetobacter sp. and Sphingomonas sp. (Liu et. al., 2009), as well as yeast, Candida tropicals (Wang and Gong, 2007), are reported to have been successfully immobilized and used for the degradation of acrylamide, phenol, and pyrene respectively. The results show that the immobilized cells have better degradation ability compared to free-cells of the microorganisms.
The used of gellan gum for encapsulation of microbial cells has been proposed from previous study by Moslemy et. al., (2002) as it has many advantages than the use of k-carrageenan and alginate. Gellan gum is produce by the microorganism, Sphingomonas alodea (ATCC 31461). Sphingomonas elodea was formerly known as Pseudomonas elodea (Donner and Douds, 1995). Gellan gum is a linear tetrasaccharide. It has high molecular weight, consisting approximately 50,000 residues and normally, it is de-esterified by alkali treatment before used in food processing industry. General chemical structure of gellan gum is presented in figure 3. Gellan gum can forms gels when there is positive charged ion (cations) available or added. Therefore, the texture and the thickness of gellan gum can be controlled depends on the addition of calcium, magnesium, potassium, and sodium salts. Gellan gum-encapsulated cell involve the production of microbeads by emulsification-internal gelation.