**3. Microbial degradation of acrylamide**

The interest in environmental problems is continuously growing and there are increasing demands to seek the sustainable and controllable process which do not burden the environ‐ ment significantly. Biodegradation is one of the classic methods for removal of undesired organic compounds to concentrations that are undetectable or below limits established as acceptable by regulatory agencies.

Acrylamide is likely to partially biodegrade in water within approximately 8-12 days [13]. If released on land, acrylamide can be expected to leach readily into the ground and biodegrade within a few weeks. In five surface soils that were moistened to field capacity, 74-94% degradation occurred in 14 days in three soils and 79 to 80% in 6 days in the other two soils [44]. Acrylamide may not be completely degraded in domestic sewage and water treatment facilities if residence times are relatively short [13, 45]. Further degradation through bioremediation of acrylamide to less harmful substances would alleviate environmental concerns.

Since 1982, microbial degradation of acrylamide has been explored extensively with a diversity of isolates (Table 1), mainly *Bacillus*, *Pseudomonas* and *Rhodococcus*[3, 46-55]. Further, numerous other microorganisms including the representatives of *Arthrobacter*, *Xanthomonas*, *Rhodopseu‐ domonas*, *Rastonia*, *Geobacillus*, and a newly family of Enterobacteriaceae [49, 56-62]. *Aspergillus oryzae*, a filamentous fungal has also been documented as an acrylamide degrader [63].

Several acrylamide degraders use a coupling reaction of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) for biotransformation of acrylonitrile to acrylic acid via acrylamide as an intermediate [46, 56]. For example, *R. rhodochrous* J1 changed acrylonitrile to acrylamide and subsequently to acrylic acid [47] and *R. erythropolis* utilized either 2-arylpropionamides or acrylamide to form acrylic acid and ammonia [64]. In China, *Nocardia* sp. 163, a soil derived bacterium from Taishan Mountain harboring the highest nitrile hydratase activity on acrylo‐ nitrile was also used frequently for bioconversion of acrylamide [65]. Another prominent example is *Rhodococcus* sp. AJ270 which is a powerful and robust nitrile hydratase/amidasecontaining microorganism isolated by Guo et al [66]. An aliphatic amidase (amidohydrolase) has been found to be the responsive enzyme for the deamidation of acrylamide to acrylic acid and ammonia [50, 59, 62, 64-67].

fresh water by bacteria with a half-life of 55-70 h, after acclimatization for 33-50 h [41]. Acrylamide has been shown to remain slightly longer in estuarine or salt than fresh water [15].

Acrylamide releases to land and water from 1987 to 1993 totaled over 18.16 tons of which about 85 percent was to water, according to Toxic Chemical Release Inventory of the U.S. Environ‐ mental Protection Agency (EPA) [40]. These releases were primarily from plastic industries which use acrylamide as a monomer. In 1992, discharges of acrylamide, reported to the Toxic Chemical Release Inventory by certain US industries included 12.71 tons to the atmosphere, 4.54 tones to surface water, 1,906.8 tones to underground injection sites, and 0.44 tones to land [4]. In an EPA study of five industrial sites that produce acrylamide and polyacrylamide, acrylamide (1.5 ppm) was found in only one sample downstream from a polyacrylamide producer and no acrylamide was detected in soil or air samples [13]. Concentrations of 0.3 ppb to 5 ppm acrylamide have been detected in terrestrial and aquatic ecosystems near industrial areas that use acrylamide and/or polyacrylamides [42-43]. Cases of human poisoning have been documented from water contaminated with acrylamide from sewer grouting. The acrylamide monomer was found to remain stable for more than 2 months in tap water [22].

Atmospheric levels around six US plants were found on an average of < 0.2 μg/m3

**3. Microbial degradation of acrylamide**

102 Applied Bioremediation - Active and Passive Approaches

acceptable by regulatory agencies.

in either vapor or particulate form [15]. The vapor phase chemical should react with photo‐ chemically produced hydroxyl radicals (half-life 6.6 h) and be washed out by rain [15].

The interest in environmental problems is continuously growing and there are increasing demands to seek the sustainable and controllable process which do not burden the environ‐ ment significantly. Biodegradation is one of the classic methods for removal of undesired organic compounds to concentrations that are undetectable or below limits established as

Acrylamide is likely to partially biodegrade in water within approximately 8-12 days [13]. If released on land, acrylamide can be expected to leach readily into the ground and biodegrade within a few weeks. In five surface soils that were moistened to field capacity, 74-94% degradation occurred in 14 days in three soils and 79 to 80% in 6 days in the other two soils [44]. Acrylamide may not be completely degraded in domestic sewage and water treatment facilities if residence times are relatively short [13, 45]. Further degradation through bioremediation of

Since 1982, microbial degradation of acrylamide has been explored extensively with a diversity of isolates (Table 1), mainly *Bacillus*, *Pseudomonas* and *Rhodococcus*[3, 46-55]. Further, numerous other microorganisms including the representatives of *Arthrobacter*, *Xanthomonas*, *Rhodopseu‐ domonas*, *Rastonia*, *Geobacillus*, and a newly family of Enterobacteriaceae [49, 56-62]. *Aspergillus oryzae*, a filamentous fungal has also been documented as an acrylamide degrader [63].

Several acrylamide degraders use a coupling reaction of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) for biotransformation of acrylonitrile to acrylic acid via acrylamide as an

acrylamide to less harmful substances would alleviate environmental concerns.

(0.007 ppb)

In 1990, Shanker and his colleagues isolated an acrylamide-degrading bacterium, *Pseudomo‐ nas* sp., from soil using an enrichment method. This bacterium degraded high concentration of acrylamide (4 g/l) to acrylic acid and ammonia. An amidase was also found to be the relevant enzyme for the hydrolysis of acrylamide and other short chain aliphatic amides like formamide and acetamide but not on acrylamide analogues, methacrylamide and *N, N*-methylene bisacrylamide [48].

Many aerobic microorganisms utilize acrylamide as their sole source of carbon and ener‐ gy including *Pseudomonas* sp. and *Xanthomonas maltophilia*. Nawaz and his team found amidase in cell free extracts of these species and suggested it was involved in acrylamide degradation [49]. This is consistent with their earlier conclusion of acrylamide degrada‐ tion by *Rhodococcus* sp. [50]. Later, the denitrifying bacteria, *Pseudomonas stutzeri* was found to use acrylamide as substrate in the acrylonitrile–butadiene–styrene resin waste‐ water treatment system. The strain could remove acrylamide at concentrations below 440 mg/l under aerobic conditions [52]. Acclimation of microorganisms is believed to enhance acrylamide biodegradation. Complete degradation of acrylamide at 10–20 ppm in river water occurred in about 12 days with non-acclimated microorganisms, but in only 2 days with acclimation [3]. In 2009, scientists in Malaysia reported two acrylamide-degrading bacteria, *Bacillus cereus* DRY135 and *Pseudomonas* sp. DRYJ7. Acrylic acid was also detect‐ ed as a metabolite in the degradation [53-54]. *Aspergillus oryzae* KBN 1010 has been the only fungi documented as an acrylamide degrader [63].

In domestic wastewater in Thailand, four novel acrylamide-degrading bacteria (*Enterobacter aerogenes*, *Kluyvera georgiana*, *Klebsiella pneumoniae*, and *Enterococcus faecalis*) were isolated. *E. aerogenes* and *K. georgiana* showed degradation potential of acrylamide up to 5000 ppm at the mesophilic temperatures and could degrade other aliphatic amides especially short to medium-chain length but not amide derivatives [60-61]. Removal of acrylamide and ammo‐ nium nitrogen from industrial wastewater by *E. aerogenes* was generally higher than that by mixed cultures of microorganisms [68].

Degradation of acrylamide under anaerobic conditions has been rarely described. Recently a new strain of *Rhodopseudomonas palustris* was found capable of using acrylamide under photoheterotrophic conditions but grew poorly under anaerobic dark or aerobic conditions. A study of acrylamide metabolism by nuclear magnetic resonance showed the rapid deami‐ dation of acrylamide to acrylate and further to propionate [57]. More recently, the denitrifying


bacterium, *Ralstonia eutropha* TDM-3 isolated from the wastewater treatment system associated with the manufacture of polyacrylonitrile fiber consumed acrylamide to concentration of 1446 mg/l, above which it was toxic [58]. This report is similar with the potential of soil bacteria, *Ralstonia eutropha* AUM-01 and *Geobacillus thermoglucosidasius* AUT-01 [59, 62]. One report, and perhaps most interesting, removal of acrylamide has been found potentially with the natural microbial populations in Rocky Ford Highline Canal, Colorado USA [69]. Degradation of acrylamide occurs under aerobic or anaerobic conditions, with nitrate serving as the most favorable anaerobic electron acceptor. Phylogenetic analysis of these cosmopolitan microor‐ ganisms suggest the potential for biodegradation in similar lotic systems such as *Pseudomo‐ nas*, *Rhodococcus*, and *Bacillus*. New proteobacterial genera (*Pectobacterium*, *Citrobacter*, *Delftia*, *Comomonas*, and *Methylobacterium*) were also found [69]. Microbial degradation of a lipid in conjunction with acrylamide was also report with *Pseudomonas aeruginosa* DS-4. Salad oil was believed to be an essential factor for acrylamide biodegradation by this bacterium. The degradation rate of acrylamide was affected by the incubation time of the acclimated strain DS-4. Longer incubation time with acrylamide resulted in more efficient degradation [55].

Removal of Acrylamide by Microorganisms http://dx.doi.org/10.5772/56150 105

Until now, we can not deny possible routes for acrylamide other than deamination via amidase [50, 59, 62, 64, 67]. The subsequent fate of acrylate is not well understood but probably involves pathways and enzymes that have been characterized to various degrees for other acrylate utilizing bacteria (Figure 1). Acrylate metabolism is believed to proceed via hydroxylation to β-hydroxypropionate, then oxidized to CO2 [48] or reduced to propionate [57]. Another plausible pathway for mineralization of acrylamide is via formation of acrylyl CoA which

A powerful tool that also enables unraveling acrylamide metabolic pathways is the sequential induction of catabolic enzymes and intermediatary metabolites. Further, insight into degra‐ dative pathways is also provided from assaying the probable key proteins that are synthesized at sufficient levels when acrylamide is present. Using proteome analysis, fifteen proteins differentially expressed from *Enterobacter aerogenes* grown on acrylamide were identified. Six protein homologues with amidohydrolase, urease accessory protein, quaternary ammonium compound resistance proteins, dipeptide transport protein, Omp36 osmoporin and large conductance mechanosensitive channel proteins (MscL) are seemingly involved in acrylamide stress response and its degradation. Five proteins identified as GroEL-like chaperonin, ArsRtranscriptional regulator, Ts- and Tu-elongation factor and trigger factor and four proteins (phosphoglycerate kinase, ATP synthase β-subunit, malate dehydrogenase and succinyl-CoA synthetase α-subunit) are expected to be relevant to adaption of *E. aerogenes*in the presence of acrylamide [70]. Based on the results, Charoenpanich and Tani have proposed acrylamide may be assimilated using Omp36 osmoporin and dipeptide transport proteins. Acrylamide is toxic, indeed lethal, to most microorganisms, however some bacteria have adapted their metabolism to use this substance as an energy source. Important to this adaptation is the evolution of genes that encode amidohydrolase (amidase) and other synthesis proteins that deaminate acryla‐

**4. Metabolism of acrylamide**

eliminates lactate as a final product [48].

**Table 1.** Acrylamide-degrading microorganisms.

bacterium, *Ralstonia eutropha* TDM-3 isolated from the wastewater treatment system associated with the manufacture of polyacrylonitrile fiber consumed acrylamide to concentration of 1446 mg/l, above which it was toxic [58]. This report is similar with the potential of soil bacteria, *Ralstonia eutropha* AUM-01 and *Geobacillus thermoglucosidasius* AUT-01 [59, 62]. One report, and perhaps most interesting, removal of acrylamide has been found potentially with the natural microbial populations in Rocky Ford Highline Canal, Colorado USA [69]. Degradation of acrylamide occurs under aerobic or anaerobic conditions, with nitrate serving as the most favorable anaerobic electron acceptor. Phylogenetic analysis of these cosmopolitan microor‐ ganisms suggest the potential for biodegradation in similar lotic systems such as *Pseudomo‐ nas*, *Rhodococcus*, and *Bacillus*. New proteobacterial genera (*Pectobacterium*, *Citrobacter*, *Delftia*, *Comomonas*, and *Methylobacterium*) were also found [69]. Microbial degradation of a lipid in conjunction with acrylamide was also report with *Pseudomonas aeruginosa* DS-4. Salad oil was believed to be an essential factor for acrylamide biodegradation by this bacterium. The degradation rate of acrylamide was affected by the incubation time of the acclimated strain DS-4. Longer incubation time with acrylamide resulted in more efficient degradation [55].
