In the process of activated sludge floc-forming microbes degrade wastewater sludge, pollutants or any organic material, under aerobic conditions, to effect a biosolid–liquid separation.
While a fraction of the organic material is used for the synthe-sis of new microorganisms, resulting in an increase in biomass, the remaining material is channeled into metabolic energy and oxidized to carbon dioxide, water, nitrates, sulphates and phos-phates to provide energy for both synthesis and cellular functions (Fig. 9). The settled biosolids are subsequently recycled to aer-ation tanks in order to maintain the biomass concentraer-ation and the supernatant is discharged. Once the organic waste material becomes exhausted then the organisms will begin endogenous respiration to oxidize cellular material. Unfortunately, a dis-advantage of an aerobic treatment is the production of large amounts of biosolids (sludge) which contains volatiles, organic solids, nutrients, pathogens, heavy metals, inorganic ions, toxic organic chemicals and the original problem of dissolved organic waste is now transformed into a problem of particulate waste.
Aerobic respiration is defined as the aerobic catabolism of nutrients to carbon dioxide and water involving glycolysis, the tricarboxylic cycle, an electron transport system and molecu-lar oxygen as the final electron acceptor: this type of aerobic digestion is notable in organisms that require molecular oxygen,
Fig. 9. Aerobic digestion of waste involving enzymes and microorganisms.
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and facultative anaerobes that are capable of aerobic respiration but can switch to fermentation if oxygen is unavailable. Typ-ical assays for enzymes associated with this aerobic digestion (dehydrogenase EC. 1.1.1; kinase EC. 2.7.1; aldolase EC. 4.1.2;
enolase EC. 4.2.1; isomerase EC. 5.3.1; mutase EC. 5.4.2, are listed;Table 2).
4.2. Anaerobic digestion
By definition anaerobic digestion is the breakdown of organic material into biogas (CH4, CO2) and in which a molecule other than oxygen is the final electron acceptor. For exam-ple, sulphate-reducing bacteria transfer electrons to sulphate (SO42−) reducing it to H2S, while others (nitrate reducers) trans-fer the electrons to nitrate (NO3−) reducing it to nitrite (NO2−), nitrous oxide (NO) or nitrogen gas (N2). Alternative electron acceptors are FeIII and MnIV. It is critical that an understand-ing of the conditions, under which anaerobic digestion is takunderstand-ing place, be made before there can be any assessment of biore-mediation potential. Amongst the many treatment technologies available, anaerobic treatment process has proved to be unique and the most beneficial stabilisation technique as it optimizes cost effectiveness, it is environmentally sound, minimises the amount of final sludge disposal and has an ability to produce a net energy gain in the form of methane gas[32]. The anaero-bic treatment technology currently available, however, is only capable of partially treating waste in a conventional wastew-ater treatment system with high levels of degradation requir-ing longer retention times and/or further treatment methods [33].
It is commonly accepted that there are four distinct steps: (a) complex organic matter is decomposed into simple soluble organic molecules using water (hydrolysis) and hydro-lase enzymes (glucosidases, lipases, proteases, sulphatases, phosphatases); (b) chemical decomposition of these single
monomeric unit molecules (monosaccharides, amino acids, fatty acids, glycerol) into volatile fatty acids by a process termed acidogenesis; (c) acetogenic fermentation into acetate, H2and CO2; (d) methanogenic conversion into CH4and CO2 (Fig. 10).
4.2.1. Hydrolysis
Wastewater treatment bioreactors are complex ecosystems that contain a wide variety of organic substances and a mixed culture of heterogeneous microbial populations that effect sequential substrate removal when complex substrates are degraded. In such mixed cultures, sulphate-reducing bacteria (SRB) are able to compete, in the presence of sulphate, with methanogenic bacteria (MB) and acetogenic bacteria (AB) for the available substrates [34–36]. Biological anaerobic wastewater treatment systems, in which complex organic matter is completely degraded by SRB are a promising alternative for the methanogenic wastewater treatment systems [37] and the complex physico-chemical sulphate removal methods [38]. In conventional methanogenic anaerobic digestion systems, sol-ubilisation rates for primary sewage sludge (PSS) are reported at about 10 days in yields of 5–10% [39–43]while enhanced degradation of 52% has resulted under sulphidogenesis [44].
There is still no consensus, however, on the mechanism of enhanced hydrolysis though the key might be the activation of the hydrolytic enzymes through a sulphide gradient generated in situ during sulphate reduction[45].
Under anaerobic conditions the simple organic electron donor molecules, e.g. lactic acid, are used by the SRBs such as Desul-fovibrio and Desulfuromonas to reduce sulphate to hydrogen sulphide and bicarbonate with a resultant increase in pH (Eq.
(8)). Soluble metal salts react with H2S in situ to produce insolu-ble metal sulphides, thereby reducing the concentration of metals and salts to acceptable levels (Eq.(9)). Bicarbonate ions react with protons to form CO2and water, thus removing the ‘acidity’
Fig. 10. Anaerobic digestion of complex organic matter into methane and carbon dioxide.
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from solution as CO2gas (Eq.(10)).
3SO42−+ lactate → 3H2S + 6HCO3− (8) H2S + M2+→ MS(s) + 2H+ (9)
HCO3−+ H+→ CO2(g) + H2O (10)
Sulphate-rich wastewater is produced by many industries such as sulphuric acid in food processes, thiosulphate in the photo-graphic industry, sulphite in tanneries, the sea-food processing industry, the leaching of sulphur rich soils in land fills and mines and by power-plant flue gases from the combustion of sulphur containing fuel[46].
Several enzymes in anaerobic sulphate reduction are worthy of mention. Adenosine 5-phosphosulphate (APS), synthesized from sulphate and ATP with the enzyme ATP sulphurylase (EC.
2.7.7,Table 2), serves as the nucleoside sulphate donor in this process, and therefore plays a crucial role in sulphate activation, the key step for sulphate utilisation[47–50]. APS is then bro-ken down into sulphite and adenosine monophosphate (AMP) by APS reductase (EC. 1.8.99,Table 2) followed by a further reduction to sulphide by sulphite reductase (EC. 1.8.99,Table 2) [51].
In an extensive study on the enzymology within an anaero-bic biosulphidogenic reactor[49,52–55], it was established that the enzymatic activities associated with sludge solubilisation and enhanced hydrolysis of complex polymeric organic carbon are found predominantly associated with the organic particulate network. Disruption of this network increases the susceptibil-ity of the macromolecules, entrapped within the floc and hence protected from enzymatic degradation, to be attacked by the hydrolytic enzymes that would ultimately lead to enhanced solubilisation of sludge. The rate at which enzymatic hydrol-ysis proceeds is best described by first order kinetics as it is rate-limiting and is strongly influenced by environmental and operational parameters such as pH, temperature, biomass, par-ticle/floc size, type and concentration of particulate substrate and production concentration and by the reaction between the enzyme and its substrate[56]. Hydrolysis of these particulate organics is also enhanced in the presence of sulphide. The lat-ter, apart from being a strong reducing agent and capable of reducing disulphide linkages that are essential for maintaining the three dimensional conformational structure, is also shown to increase the specific activity of all hydrolytic enzymes by nearly 5–10-fold[52–55]. Since the biopolymers (proteins, car-bohydrates and lipids) are the major particulate organic fractions in sludge[57–59]the activities of the ubiquitous enzymes like lipases (EC. 3.1.1,Table 2), phosphatases (EC. 3.1.3,Table 2), sulphatases (EC. 3.1.6,Table 2),␣-, -glucosidases and cellu-lases (EC. 3.2.1, Table 2), and proteases/peptidases (EC. 3.4, Table 2), are equally important in the hydrolysis process. It is proposed that the products of biological sulphate reduction both directly and indirectly facilitate the contact between enzyme and substrate thereby enhancing overall enzyme activity. This is due to the neutralisation of the ions on the floc surface by sul-phide, sulphite and associated bicarbonate and hydroxide ions, destroying the overall integrity of the floc structure and exposing
more substrate for enhanced enzyme activity[60]. Alternatively, the sulphite and sulphide species liberated during the sulphate reduction process, interact directly with the enzymes on the floc surface thereby enhancing their activity.
The initial development of anaerobic treatment processes, over a century ago, was for the treatment of domestic wastewa-ters, using anaerobic filters and hybrid processes that are still of interest today. Its applications then expanded to include separate sludge digestion and then to industrial wastewater. Biological stabilisation is widely considered to be one of the most attractive methods of reducing the major portion of the organic fraction in sludge, and anaerobic processes are favoured over aerobic digestion owing to the cost of aeration, the ability of anaerobic systems to maintain their temperature and the value of methane as a renewable energy source.
4.2.2. Acidogenesis
During this phase simple monosaccharides are converted via a series of acidogenic enzymes and the glycolytic pathway to ATP, NADH and pyruvate which is regarded as a key metabolite in anaerobic digestion. It is metabolized into acetyl CoA and formate through formate C-acetyltransferase (pyruvate formate lyase) (EC. 2.3.1.54,Table 2)[61,62]or into lactate via reduction with lactate dehydrogenase (EC. 1.1.1,Table 2). In butanediol fermentation, two pyruvates are condensed with acetolactate synthase (EC. 2.2.1.6,Table 2) [63]to yield CO2 and aceto-lactate which is decarboxylated to acetoin (EC. 4.1.1.5,Table 2) [64]and eventually to 2,3-butanediol via a dehydrogenase[65].
Formate is converted by formate hydrogen lyase [66] to H2 plus CO2while acetyl CoA is either reduced to acetaldehyde via acetaldehyde dehydrogenase and then to ethanol via alco-hol dehydrogenase or converted to acetate via the high energy acetyl phosphate. Alternatively, two acetyl CoA molecules may condense producing acetoacetyl-CoA that can lose coenzyme A and CO2to form acetone [67]. An initial transcarboxylase (EC. 2.1.3.1,Table 2) reaction with methylmalonyl SCoA results in a conversion of pyruvate into oxaloacetate with a concomi-tant release of propionyl CoA. The final step is the release of propionic acid when CoASH is transferred to succinate [68].
Succinate can also be converted to succinyl-SCoA, and then back to methylmalonyl-SCoA via methylmalonyl-SCoA mutase, a Vitamin B12containing enzyme[69].
After an initial activation as their coenzyme A derivatives the long chain fatty acids are enzymatically degraded by sequen-tial removal of two-carbon units liberating, after each cycle, acetyl CoA, which enters the metabolic pool and a fatty acid with two carbons less (EC. 2.3.1.16,Table 2). Propionyl CoA is the ultimate product in the case of a fatty acid with odd-numbered carbon atoms.
It is not the intention within the current article to give an in-depth analysis of anaerobic amino acid degradation and readers are directed to two excellent reviews[70,71]. All twenty amino acids are first enzymatically deaminated, via respective transam-inases (EC. 2.6.1,Table 2), before their carbon skeletons are sub-sequently degraded into seven different metabolites—pyruvate, acetyl CoA, acetoacetyl-CoA,␣-ketoglutarate, propionyl CoA, fumarate and oxaloacetate.
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4.2.3. Acetogenesis
Acetogenic enzymes convert the products of the acidogenic step into acetate, H2and CO2that ultimately are the substrates for the methanogenic breakdown.
4.2.4. Methanogenesis
This constitutes the enzymatic production of methane from acetate or from a reduction of CO2(Eqs.(11)and(12)). Molec-ular hydrogen or reduced coenzyme F420are the electron donors with mixed disulphides (coenzymes M and B) as electron accep-tors. Eight enzymes and a formyl, methenyl and methyl tetrahy-dromethanopterin are involved in the reduction of CO2[72].
CO2+ 4H2→ CH4+ 2H2O (11)
CH3COO– → CH4+ CO2 (12)
Initially, methanofuran interacts with CO2 with assistance of formylmethanofuran dehydrogenase (EC. 1.2.99, Table 2) [73] to produce formylmethanofuran. Coupled with tetrahy-dromethanopterin and a series of enzymes involving a trans-ferase, a cyclo-hydrolase (EC.3.5.4,Table 2) a dehydrogenase and a reductase (EC.1.5.99,Table 2)[74–78]the C1unit is car-ried, via coenzyme F420 into methyltetrahydromethanopterin.
In the final step, the C1 unit is associated with coenzyme M and a transferase enzyme (EC. 2.1.1.86, Table 2) before being reduced to methane [79]. Coenzyme B (7-mercapto-heptanoylthreoninephosphate) reacts with methyl coenzyme M liberating methane and coenzyme-M-S-S-heptanoylthreonine phosphate under the influence of methyl reductase (EC. 2.8.4.1, Table 2)[80].
A slight variation is reported[81]for the anaerobic enzymatic conversion of acetate to methane (Eq.(12)). The acetate, that is first activated as its coenzyme A derivative, is oxidised to CO2 with carbon monoxide dehydrogenase while its methyl group is transferred via tetrahydrosarcinapterin to coenzyme M. This methylcoenzyme M is reductively demethylated with coenzyme B to methane and the resulting heterodisulphides converted back to their sulphydryl forms.
4.3. Bioremediation/biodegradation
Polycyclic aromatic hydrocarbons (PAHs), widely dis-tributed in the environment mainly from anthropogenic activities, are organic chemicals that are cytotoxic, mutagenic and carcinogenic. Aerobic degradation of PAHs is a benign process that involves the oxidation of the aromatic ring by specific dioxygenases and a complete biotransformation into carbon dioxide and water. Naphthalene, one of the simplest PAHs, serves as model for understanding their biodegradation.
Naphthalene-1,2-dioxygenase (EC. 1.14.12,Table 2) catalyses the oxidation, with molecular oxygen, of naphthalene to cis-1,2-dihydroxy 1,2-dihydronaphthalene[82]while salicylate hydroxylase (monooxygenase) (EC.1.14.13, Table 2) oxida-tively decarboxylates salicylate to catechol and carbon dioxide [83–85]. Both of these enzymes are assayed by measuring the decrease in absorbance at 340 nm due to the consumption of NADH in the presence of substrates.
All of the BTEX compounds (benzene, toluene, ethylben-zene and xylene) as well as the polychlorobenethylben-zenes (PCBs) have at least one aerobic pathway that includes degradation to a substituted catechol. Benzene is degraded to catechol; toluene has many separate biodegradative pathways, some of which include 3-methylcatechol as an intermediate product; ethylben-zene is degraded to 3-ethylcatechol; xylenes are all metabo-lized to mono-methylated catechols and the chlorobenzenes to chlorocatechol. In each of these cases, the aromatic ring of the substituted catechol is later cleaved by either an intradiol (ortho-cleaving) (EC.1.13.11.1, Table 2) catechol 1,2 dioxygenase or extradiol (metacleaving) catechol 2,3-dioxygenase enzyme (EC.1.13.11.2,Table 2). Subsequent metabolic pathways lead to acetaldehyde, acetate, succinate, pyruvate and eventually CO2 and H2O.
It has recently been reported[86]that sulphate reducers are capable of degrading a wide variety of PAHs by modification of the aromatic ring—in particular demethylation, dehalogenation, reduction, removal of amino, hydroxyl and methoxyl groups as these processes detoxify potential xenobiotics [87–92]. In all of these enzymatic degradative pathways, the intermedi-ate is an aromatic carboxylintermedi-ate coenzyme A thioester, formed through a benzoate CoA ligase (EC. 6.2.1.25,Table 2), lead-ing to rlead-ing reduction, hydration, dehydrogenation, rlead-ing cleavage to 3-hydroxypimelyl CoA and eventually to acetyl CoA[93].
Naphthalene, toluene, ethylbenzene and xylene follow similar pathways initially involving a carboxylation of the aromatic ring while with the biodegradation of halobenzenes a series of dehalogenations with dehalogenases (EC. 3.8.1,Table 2) take place.
To date (2004), there are over 1000 enzymes reported to be involved in the biodegradation of aromatic systems, whether they be organic pollutants or not and it is beyond the scope of this paper to explore this any further. A description of all of these alternate pathways has appeared[94].
4.4. Biopulping/biobleaching
Biopulping is the treatment of lignocellulosic materials with oxidative lignin-degrading fungal enzymes prior to thermome-chanical pulping. This biological process is reputed to increase paper-strength and reduce both chemical energy consump-tion and environmental impact. These enzymes, which include lignin and manganese peroxidase and laccase, are respon-sible for the oxidative biodegradation of PAHs (anthracene, benzo[a]pyrene) [95–101] into CO2 and H2O. Lignin per-oxidase (LiP) (EC.1.11.1, Table 2) use hydrogen peroxide to catalyze one-electron oxidations of phenolic and non-phenolic compounds leading to alkyl aryl cleavage, aromatic ring cleavage, demethylation, hydroxylation and polymeriza-tion while manganese peroxidase (MnP) (EC.1.11.1, Table 2) catalyze the Mn-mediated oxidation of lignin and phenolic compounds.
Laccases [p-diphenol-dioxygen oxidoreductase] (EC.1.10.
3.2,Table 2) belong to the group of enzymes called blue copper oxidases that catalytically oxidise phenols[102]or chlorinated biphenyls with a four-electron reduction of O2to H2O. Artificial
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substrates such as ABTS (2,2 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid), HBT (hydroxyl benzotriazole) or violuric acid can act as mediators enabling the oxidation of non-phenolic compounds which cannot be oxidized by laccases on their own, thereby expanding the range of applications of these enzymes [102].
It has been realized that thermostable xylanases can be implemented in biobleaching. [103] The process of lignin removal from chemical pulps to produce bright or completely white finished pulp is called ‘bleaching.’ Present-day bleaching of kraft pulp uses large amounts of chlorine-based chemicals and sodium hydrosulfite that produce toxic, mutagenic, persis-tent chemicals that bioaccumulate in biological systems. The main enzyme needed to enhance the delignification of kraft pulp is endo-xylanase, but enrichment with other enzymes such as mannanase, galactosidase, arabinosidase, glucosidase and xylosidase have been shown to improve the effect of enzymatic treatment of kraft pulp[104–107]. The enzyme does not bleach pulp, but rather changes the pulp structure thereby opening it up for further degradation. The cleavage of the carbohydrate portion of lignin-carbohydrate complex to produce smaller residual lignin molecules, which are easier to remove, is also a possible mechanism of xylanase prebleaching.
The decolourisation of textile dyes may also be regarded as a biobleaching process and the biological enzymatic treatment of industrial wastewater dyes remains one of the most chal-lenging. Extensive studies are forthcoming on dye decolouri-sation by non-specific extracellular oxidative enzymes (lignin peroxidase, manganese peroxidase, copper-containing phenol oxidase) from white-rot fungi[108–113]though these all have limited potential in both efficiency and reliability. Aromatic azo-dyes, in particular, are environmentally toxic and muta-genic and their enzymatic degradation involves the anaero-bic reduction of the azo bond ( N N ), with azo-reductases followed by an aerobic biotransformation of the generated aromatic amines into CO2, NH3 and H2O. Mechanistically azo reductases (EC.1.7.1.6,Table 2) which may be classified as flavoenzymes [114], may either transfer reducing equiva-lents such as NAD(P)H to the azo dye directly or through redox mediators (RM) like methyl viologen, menadione, neu-tral red, janus green[115], anthroquinone-2-sulphonate (AQS) or anthroquinone-2,6-disulphonate (AQD) (EC.1.7.1.6,Table 2) [116].
4.5. Bioleaching
This is described as the extraction of metals from their respec-tive ores by biotechnological processes and enzyme based tech-nologies for metal removal present an economic alternative for today’s mining, mineral and waste water treatment industries.
The role of enzymes in a bioleaching process may be asso-ciated with either: (i) active transport ATPases, e.g. cadmium, copper, arsenate or (ii) direct enzymatic removal, e.g. mercury, iron, chromate[117,118]. With respect to the former the best characterized system is that for the essential metal ion Cu(II) and its concentration in the cell is delicately balanced by means of CopA, CopB, CopC, CopD, CopY and CopZ ATPases[119].
Fig. 11. Copper transport in Pseudomonas syringae[119].
Extracellular copper in the form of Cu(II) reaches the periplas-mic space (Fig. 11) through the porous outer membrane assisted by CopA. A second ATPase, CopC, transports the copper to the inner membrane and to CopD that allows the copper to enter the cytoplasm. Here the Cu(II) is reduced to Cu(I) via a NADH-cupric reductase system. Within the periplasm are two blue copper-binding proteins—CopY (repressor) and CopZ (chap-erone) that sense levels of copper and switch off (or on) the respective operons. This allows for the export of Cu(I) through CopB[120,121]. The assay for cupric reductase is reported[122]
(EC.1.16.1.1,Table 2).
Enzymatic processes, for the removal of metal ions from sludge waste effluent streams, use metal-desolubilising ligands on a continuous basis with extracellular or intracellular depo-sition of metals as a metal-ligand precipitate. Metal reduction usually results in the precipitation of low valence, reduced, forms of metals, and has therefore been proposed as a strat-egy to treat water contaminated with a range of metals and radionuclides. Mercury is a model example of an enzymatic removal process as it lends itself to inactivating essential thi-ols that are part of enzymes and proteins[123]. Some bacteria (Staphylococcus, Bacillus, Escherichia, Pseudomonas, Serra-tia, Thiobacillus) contain a set of genes that form a Hg(II) (mer) resistance operon which not only encodes for the production of a periplasmic metal binding enzyme that collects Hg(II) from the surrounding environment but also for a membrane associ-ated transport enzyme that carries the Hg to the cytoplasm for removal. The first, organomercurial lyase (EC.4.99.1.2,Table 2)
Enzymatic processes, for the removal of metal ions from sludge waste effluent streams, use metal-desolubilising ligands on a continuous basis with extracellular or intracellular depo-sition of metals as a metal-ligand precipitate. Metal reduction usually results in the precipitation of low valence, reduced, forms of metals, and has therefore been proposed as a strat-egy to treat water contaminated with a range of metals and radionuclides. Mercury is a model example of an enzymatic removal process as it lends itself to inactivating essential thi-ols that are part of enzymes and proteins[123]. Some bacteria (Staphylococcus, Bacillus, Escherichia, Pseudomonas, Serra-tia, Thiobacillus) contain a set of genes that form a Hg(II) (mer) resistance operon which not only encodes for the production of a periplasmic metal binding enzyme that collects Hg(II) from the surrounding environment but also for a membrane associ-ated transport enzyme that carries the Hg to the cytoplasm for removal. The first, organomercurial lyase (EC.4.99.1.2,Table 2)