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Enzymes in biological wastewater treatment

What are enzymes?


Enzymes are compounds, that enable and facilitate the processes of biochemical reactions. Without enzymes:

Under normal conditions (for example temperature, pressure, low concentration of reactive components) all these biochemical reaction would not even run. eactive speed would be extremely low !e"ative effects on the reaction would show stron"ly Uncontrolled ne"ative effects accompanyin" the reaction li#e uncontrolled increase in temperature, or chan"es in $%, etc...

Enzymes have similar function as catalyst durin" chemical reactions and that is why they are sometimes re"arded as biocatalysts. Enzymes are or"anic protein substances. Enzymes are divided in to two "roups dependin" on the structure of the resultin" molecule: Proteinic enzymes (sin"le component) are composed of only proteins. &mino acids create an active centre on the protein and that defines specificity of the enzyme (the type of reaction of substances which the enzyme will be part of). 'o the "roup of sin"le component enzymes we include hydrolase, enzymes catalysin" the process of hydrolase which is the fracturin" of complex substances into ones with simpler molecules. 'he basic hydrolytic reactions durin" removal of waste waters pollution.

%ydrolase of poly and oli"osaccharides into monosaccharides %ydrolase of proteins and peptides (raction of fats into "lycerol and relevant fatty acids

Proteidic enzymes (double component) have more complicated structure because they are created from protein molecule,i.e. apoenzyme and from low molecular compound which is not a protein character,i.e. coenzyme. 'his coenzyme re"arded as cofactor plays the role of the activatin" centre

in double component enzymes. 'he chemical reaction chan"in" the substrate is happenin" ri"ht at a particular place of the coenzyme. 'he protein of the enzyme ( i.e. apoenzyme) is a"ain very

important for the specificity of enzyme, which is reflected by the fact, that enzyme effects only a specific substance and from all the possible reactions that this substance can cause, it catalyses only some. )n the other hand, the specificity of the effect of the enzyme ( i.e. type of reaction) is determined by coenzyme. *oenzymes are connected with proteins (apoenzymes) either by unstable adsorbent bond, ion bond or stable covalent bond. +t belon"s to main coenzymes, nucleotides, nucleotidphosphate, porphyrin coenzymes, coenzymes containin" vitamins and etc.

Division of enzymes
'here are thousands of enzymes that transmit biochemical reactions in botanical and animal world, and they can create many different combinations of apoenzymes and coenzymes. &ccordin" to their main effect we classify enzymes in , different cate"ories: 1. group: !ydoredu"tase

Enzymes that catalyse - oxid.reducin" reactions in cells. /ainly enzymes connected to respiratory chain and to electron transport system, for example cytochromoxidase, cytochrome, nitrate reductase which are very important for biolo"ical denitrification etc ... #. group: $ransferase Enzymes that catalyse transfer "roups of substances from donor to acceptor. (or example transaminases catalyse transfer of amino "roups durin" the metabolic action. %. group: &ydrolase Enzymes catalysin" hydrolytic reactions. +t is a hu"e complex of very important reactions durin" the start faze of or"anic pollution removal from waste water. )ri"inal substance brea#s into its components while catalysed water molecules are in action, which can be "enerally illustrated in this formula:

'hese enzymes belon" often with extracellular enzymes, i.e. they act also outside their own cells. While hydrolase in action, bi" cells brea# into small fra"ments, that can be transported into cells by transportive enzymes for further metabolism.

'. group: (yases Enzymes from this "roup also brea# chemical bonds, but not throu"h hydrolytic process but directly by fraction of bond between atoms of carbon *.* ).group: *somerases 'hese enzymes catalyse complicated chan"es inside the molecule that lead to transformations from cis - to trans - position and vice versa ( cis. and trans. conformeria, isomers) +.group: (igases 'his "roup of enzymes catalyse reaction leadin" to synthesis of more complex molecules, where the ener"y released from the ener"y trasporter &'$ (adenosine triphosphate) is re0uired. 'he synthesis with the use of ener"y can be represented as follows:

We already mentioned that enzymes can be divided into e!tracellular and intracellular accordin" to localisation, where the catalysation ta#es place. (urther division of enzymes describes if the specific enzyme in the cell is still in active form (constitutive enzyme) or if the cell has to synthesize the enzyme before the specific reaction (inductive enzyme). 'he best #nown inductive enzyme in biolo"ical waste water treatment is nitratreductase.

,unctioning of enzymes
'he first function of enzymes is initiation of reaction. 'he minimum amount of molecules of reactin" substances is re0uired for the reaction to ta#e place. When these molecules meet, then to launch the reaction you need specific amount of ener"y. We call it activating energy and the 0uantity of this activatin" ener"y depends on surroundin" conditions, specifically temperature. 'he process of biochemical reactions is mostly limited by temperature, because mainly hi"h temperatures can dama"e reactin" elements. &t temperature around 12 de"rees *elsius, which is normal for waste

water, the activatin" ener"y of many reactions is so hi"h, that the reaction would not be at all. 3ue to catalytic effect of enzymes, this activatin" ener"y can be decreased and enzymes ma#e this reaction possible.

4esides function for decrease of activatin" ener"y we can summarize the effect enzymes as follows:

Enzymes act in minimal concentrations, which are on borderline of threshold concentration 5i#e catalysts, enzymes appear unchan"ed from the reaction and not used (that does not mean that enzymes remain in medium in unchan"ed concentration, because they are protein based, they naturally decompose due to bacteria activity)

3ecrease of activatin" ener"y !on catalysed reaction *atalysed reaction

Enzymes do not have any effect on balanced position, that chemical reaction fall into (the chan"e of thermodynamic ener"y 6 is the same in both non catalysed and in catalysed reaction) Enzymes provide and accelerate reactions, but do not chan"e the balanced position to either substrate side or product side. /any enzymes start actin" when influenced by other substance, activator. &ctivators, besides chemical substances can also be factors li#e temperature, p%, oxidation reduction potential, physiochemical parameters of surroundin"s (medium)

$he process of enzymatic reaction


3urin" the biolo"ical treatment of waste water, the substrate has to under"o some enzymatic steps before it transforms itself into final product (for example residues of indecomposable pollution or biomass). Each step is catalysed by enzyme alone, so durin" the process of removal of the specific substrate, there can be hundreds and more enzymes active. 'he reason for the hi"h number of re0uired enzymes is their specificity. 'his is due to three.dimensional structure of the enzyme, in which is situated the active centre. 'he followin" picture describes connection of substrate to active area of specific enzyme. 'he base of this reaction is the creation of the comple! enzyme substrate.

Complex enzyme substrate, explanation of substrate specificity of enzymes &s seen from the followin" dia"ram, complex enzyme. substrate is in balance between ori"inal and final products. (or the balance disturbance the rules of chemical #inetics apply.

or:

eaction leadin" to creation of complex enzyme - substrate can be disturbed by various chemical or physiochemical factors, which are "enerally called *nhibition. 4efore a possible synthetic application of enzymes into the system, chec# if there are not any inhibitory factors that would act a"ainst enzymatic reactions. &s flows from the theory of enzymatic reactions, ma7ority or biochemical reactions that ta#e place in limited conditions (mainly in low temperature) have to be catalysed by these biocatalysts, otherwise they would not pro"ress at acceptable speed. /icrobial consortia used durin" biolo"ical treatment of waste water are spontaneously formed, lon" adapted to specific outer conditions, i.e. mainly to temperature and chemical content of the media - waste water. 'hat is why under normal conditions the variety and concentration of enzymes should be produced by a such consortia as is for example activated slud"e. +f not, causes can be seen in the followin" factors or combination of these factors:

/icroor"anism producin" specific enzymes are not present in consortia. 'he reasons can be unsuitable conditions for promotin" their "rowth, for example low temperature, extreme p%, lac# of oxy"en or insufficient a"e of the biomass(slud"e) which causes floodin" of these microor"anisms

8top or speed reduction of the reaction due to inhibitin" substances (mostly in industrial wastewater) *oncentration of produced enzymes is insufficient towards the concentration of processed substrate. &ll the active areas of enzymes are busy and the speed of enzymatic reaction is at maximum.

8pecific substrate can appear in wastewater only spasmodically with a short period of existence, which can cause problems with the effect of inductive enzymes

+n these cases it is sensible to thin# of artificial addition of those enzymes, whose concentration is for various reasons insufficient. e0uires carefully suspendin" inhibitory effects. &lthou"h the theory of

inhibition says that the level of inhibition can be decreased by concentration or action of specific enzyme. +n ma7ority of practical cases the dosin" of enzymes into inhibited biolo"ical system would be pointless and economically un7ustifiable.

+n wastewater treatment history can be considered as successful dosin" of followin" types of enzymes leadin" to solution of practical operational problems. *mprovement in nitrification of ammonia nitrogen )ne of the most sensitive processes durin" current biolo"ical treatment of wastewaters is biolo"ical oxidation of ammonia nitro"en to nitro"en nitrate. nitrification. 3osin" of specific enzymes that promote certain step of transfer of electrons from ammonia nitro"en to dissolved oxy"en. $rocess of nitrification is also often limited by insufficient concentration of dissolved oxy"en in biomass, that is below the saturation concentration level for oxy"en. +n this case, specific enzymes that promote the speed of the electron acceptor could help with easier transfer of the oxy"en into biomass. .esults of filamentous activated sludge bul"ing +n consortia of activated slud"e we can find many microor"anisms, that have filamentous morpholo"y. $resence of these microor"anisms in this form cause decrease in sedimentation and9. properties of activated slud"e (effect #nown as filamentous activated slud"e bul#in"). /a7ority of filamentous microor"anisms causin" this problem prefer as substrates simple saccharides, which can be products of polysaccharide decomposition. $roblems happen when products of this decomposition are released into the media in hu"e aeration tan#s and not in compartments of contact zones that are put into activatin" systems to prevent filamentous bul#in". 3ecomposition acceleration of polymer compounds, and utilization of the products from this decomposition in the contact zone can be achieved by addin" specific splittin" enzyme, for example amylase. .eduction of issues with creation of biological foam 'he problem of increased production of biolo"ical foams is the current issue of our wastewater treatment plants in the last :; years. 'he systematic research executed in the *zech republic on <8*%' $raha with the collaboration of the prominent international research centres confirmed that the immediate cause of creation of biolo"ical foam is a"ain presence of filamentous microor"anisms, but other types this time. 'his time it is a type of actinomycet filament characterized as follows:

%i"h hydrophobicity of the cell surface of filament *reation of biolo"ical surface. active substances, that stabilize biolo"ical foam %i"h affinity of substrates from "reases and oils or of the immediate products of their fraction, i.e. lon".chain fatty acids

3ominant part of other activated slud"e microor"anisms (fla#e creatin") is not able to utilize "reases or lon".chain fatty acids directly. 6rowth promotion of these fla#e creatin" microor"anism (at the expense of those foam creatin") can be achieved by addin" enzymes that accelerate hydrolysis of fat (disruptin" "lycerol bond - lon" -chain acid *:,.*:=). 'hey also promote fraction of *.* bonds and

conse0uently decomposition of lon". chain fatty acids into volatile fatty acids with carbon chains *1. *,. &ddition of these enzymes would contribute to re0uired selection by complicatin" access of unwanted microor"anism to these specific substrates. 4iolo"ical foams and poorly sedimented slud"e are very difficult to separate from purified wastewater in settlement tan#s and they contribute to increased production of suspended solids. 3ue to their biolo"ical nature they dramatically increase the indicator of residual contamination, which is accordin" to our le"islation on wastewater dischar"e, char"ed Possibility of increased biodegradability of refractory substances Waste water from biolo"ical waste water treatment plants contains refractor so called residual or"anic substances, that come from partially from the portion of the poorly biode"radable or"anic waste water pollution and partially from the microor"anic metabolic production (that create biolo"ical consortia in WW'$). 'hese substances create portion of residual contamination and contribute to increased pollution char"es. 'here is a lar"e number of causes for poor biode"radation, however one of them can be also poor concentration of enzymes for example from the "roup of lyases, that initiate fraction on or"anic bonds *.*. &lthou"h even small concentration of these naturally "enerated enzymes does not have to be sufficient for reaction initiation, due to a small number of active areas on enzymes and conse0uently to small number of interactions between these active areas and molecules of refractors presented in relatively small concentrations (due to substrate concentration in waste water). +ncreasin" the concentration of these enzymes will increase probability on interactions and can initiate fractions of substances of this type. 3osin" of these enzymes is the result of economic balance between the costs of dosa"e and possible savin"s on pollution char"es *%8>*r parameter. *? @ E! @ U @ 3E

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