Chemistry FIA3 Final - IA3 research task PDF

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Research investigation

Claim Catalysts and enzymes are the future for the manufacturing industry

Rationale In the garment manufacturing industry, especially the leather industry, the use of synthetic dyes is very common, specifically azo dyes. It is a prominent class of wastewater contaminants; hence it poses a significant threat to the environment. A large amount of wastewater containing a mixture of skin biologicals, a large variety of organic and inorganic chemicals are generated during the skin-toleather conversion process. Wastewater from tanneries classically contains high concentrations of dyes because a significant amount of wastewater contains high concentrations of azo dyes and a mix of leather biologicals (Murugananthan et al. 2004). The chemical components of azo dye (figure 1) are mainly phenolic compounds, and these dye molecules are toxic and difficult to degrade within the typical wastewater treatment system (Sadhanandam et al, 2013). Thus, there are a variety of physio- chemical treatments available to decolourise the dye-house wastewater such as precipitation, adsorption, etc. Howerver, each has inherent disadvantages including the demand for a external reagent, large sludge generaction, and are expensive and tedious (Nurhaslina et al, 2014). Therefore, in recent years, the study of the alternative of enzymatic treatment for decolorising the dye house wastewater has gianed popularity in order to reduce the cost of decolorising the dye wastewater and to improve decolourising efficiency (Kiro Mojsov et al, 2016). Enzymatic treatment systems are simpler and easy to operate in comparision to the physio-chemical treatment systems because the enzyme-catalysed reaction is more effient, selective, higher reaction rate, and milder reaction conditions (Palmieri G, Cennamo G, Sannia G, 2005). The enzyme that is applied for decolouration and degradation in industrial effluents is oxidative enzyme such as lignin peroxidase, manganese peroxidase, soybean peroxidase, horseradish peroxidase (HRP) and laccase, etc, (Mohan et al, 2005). Horseradish peroxidase (HRP) is known for its ability to efficiently cleave aromatic azo compounds in the presence of H2O2 and to breakdown and precipitate azo dyes in the industry (Mithat et al, 2012). HRP enzyme is the haemoproteins that use H2O2 as electron acceptors to catalyse the oxidation of substrates (Mithat et al, 2012); hence, when HRP enzyme react with H2O2 can produce oxidative breakdown of synthetic azo dyes. Further research has confirmed that choosing enzyme-specific encapsulation materials and optimising process conditions remain a key concern (Mohan et al, 2005). Various parameters such as contact time and H2O2 have been investigated in order to optimise the treatment condition. Furthermore, to demonstrate the application capability and advantage of which HRP enzyme for industry, the study from (Sadhanandam et al, 2013) have compared the reaction rate of free HRP and immobilised HRP enzyme base on reaction time and H2O2 concentration in the decolourising. The enzyme found in plant roots, cellulolytic bacteria, and fungi that secrete free HRP enzymes is dependent on the hydrolysis of lignocellulose into usable sugars by enzymes with specific substrate specificities (Peng Li et al, 2018). Immobilised HRP enzyme is the free HRP enzyme that has been immobilised in calcium alginate beads while retaining its full activity or most of its activity.

Therefore, the research question was developed and refined to consider the effect of the treatment conditions (contact time and H2O2 concentration) have on the rate of reaction of HRP enzyme (free and immobilised) in the dye removal. To determine whether a free HRP or immobilised HRP enzyme is effectual for industrial applications, it is essential to understand how rate of reaction of HRP enzyme (free and immobilised) affected the colour removal is based on their contact time and hydrogen peroxide concentration (H2O2).

The research question: Is immobilised horseradish peroxide (HRP) enzyme more effective than free horseradish peroxide (HRP) enzyme based on the reaction with hydrogen peroxide (H2O2) concentration and contact time in the degradation of azo dye from tannery effluent? This review will use a study from Sadhanandam Preethi, Ayyappan Anumary, Meiyazhagan Ashokkumar and Palanisamy Thanikaivelan have been undertaken to classify free HRP enzyme compared to immobilised HRP enzyme. The results of these studies will be compiled to investigate whether a free HRP or immobilised HRP catalysed is effectual for industrial applications.

Background Horseradish peroxidase (HRP) is an efficient environmentally friendly enzyme which found in the roots of horseradish plants. Figure 2 displays the structure of horseradish peroxidase, which has two metal centres, one of iron heme group and two calcium atoms. Iron has two open bonding sites, one of which is a histidine (enzyme) attached to the heme group in the proximal histidine residue (His170), which is beneath the heme group. In the resting state, the second histidine residue above the heme group on the distal side of the heme group is vacant. This site is available for hydrogen peroxide to bind to during reduction-oxidation reactions (Horseradis Peroxidase, 2020). Many researchers have demonstrated that phenolic compounds in azo dye can be degraded or decoloured by peroxidase, which has revealed that HRP plays an important role in the removal of dyes in wastewater (Mohan et al, 2005). Due to its advantages such as high activity and selectivity, high resistance to inhibition by various compounds over a wide concentration range, high operability and reliability in a variety of treatment conditions (Sadhanandam et al, 2013). HRP enzyme has been greatly used in effluent treatments as it advances long lifetime, stability, and retention of the enzymatic activity over a broad range of H2O2 concentration and short amount of time in the dye degradation. Many researchers claimed that both free and immobilised HRP enzymes were effective at the degradation of dye and immobilised HRP enzyme may provide a better environment for the enzyme activity (S Venkata Mohan et al, 2005). For the industrial application, the stability, long lifetime and cyclability of HRP enzyme in its free and immobilised forms in the reaction of azo dye degradation are critical.

Analysis and interpretation A study of (Sadhanandam et al, 2013) on the reaction of free HRP enzyme and immobilised HRP enzyme in terms of contact time and H2O2 concentration for colour removal. For all test series, each reaction mixture contains 3mL of 30mg/L dye and 0.08 U HRP. The contact time (0, 15, 30, 45, 60, 75, 90, 105, 120 min) and the concentration of H2O2 (2, 4, 6, 8, 12, 14, 16, 20 L) were varied to examine the reaction rate of the HRP. A study was carried out in triplicate and the mean was determined as the percentage of dye degradation. The mean results are displayed along with the error bars. The study had immobilised the free HRP in calcium alginate beads and was carried out to optimise the process parameters for the application of immobilized HRP for decolorizing of azo dye.

The rate of reaction of HRP enzyme during contact time in azo dye degradation

Graph 1 displays the percentage of dye degradation efficiency in the contact time of free HRP enzyme. The degradation of dye increases significantly in the first 20 min of contact time and continues until 45 min. After 45 min, there is no considerable improvement in the degradation of dye. Dye removal efficiency in wastewater using free HRP enzyme requires 45 min in contact time. Thus, it is demonstrating that free HRP has maximum activity in 45 min in order to degrade the azo dye in wastewater. Whereas the reaction rate of immobilised HRP enzyme gradually increases as contact time increases in the degradation of azo dye. To efficiently degrade the azo dye, the immobilised HRP enzyme requires a long contact time (240 min). The study has found that entrapment of HRP in calcium alginate beads while immobilising the free HRP enzyme influenced dye degradation efficiency (Sadhanandam et al, 2013). Overall, the graphs show that the azo dye degradation efficiency using free HRP has a faster reaction time (40 min) than using immobilised HRP (240 min). Limitation of evidence: Although two graphs of (Sadhanandam et al, 2013) demonstrated the reaction rate of free HRP and immobilised HRP enzyme in terms of contact time in the dye degradation, the study did not mention the definition of the free HRP and immobilised HRP enzyme. Also, the study did not include how the enzyme was recovered.

The effect of the H2O2 concentration on the rate of reaction of HRP enzymes in azo dye degradation.

Graph 3 shows that at low H2O2 concentration, the percentage of the dye degradation is approximately 42%. When the H2O2 concentration is increased, the reaction of the free HRP enzyme increases lead to the percentage of dye degradation increases to 78%. There is no significant increase after 14 μL H2O2. Hence, free HRP enzyme works most efficiency at 14 μL H2O2 in the dye degradation. Graph 4 shows that at low H2O2 concentration (4 μL), the reaction rate of immobilised HRP is higher than the reaction rate of free HRP in the percentage of dye degradation. However, at 14 μL H2O2, the reaction rate of free HRP is greater than the reaction rate of immobilised HRP. In additional, there are not substantially differences in the trends of both graphs 3 and 4. Thus, it determines whether free HRP or immobilised HRP is more effective in term of reacting with H2O2 concentration is complicated. The reuse of the enzyme is required for industrial application. However, in this study, free HRP did not have recyclability, whereas immobilised HRP did. The study of (S Venkata Mohan et al, 2005) found that immobilised enzymes have the advantage of being recyclable in the process, which reduces treatment costs.

Graph 5 depicts that the immobilised HRP enzyme can be reused for at least 3 cycles without losing much of its activity in terms of contact time. The immobilised HRP has been removing colours in

accordance with identical patterns for all three cycles. There would appear to be a modest decline in decolourisation throughout the second and third cycles of the experiment. The study therefore shows that the immobilised HRP can lower colour removal costs for industrial applications. Clearly, free HRP has a faster reaction than immobilized HRP in terms of contact time in the colour removal in waster; however, in term of react with H2O2 concentration, the reaction rate of free HRP and immobilised HRP are similar which is not demonstrated that the immobilised HRP is more effective than the free HRP enzyme. Immobilised HRP can be reused for at least 3 times whereas free HPR cannot, which demonstrates application capability and advantage of immobilised HRP at industrial, particularly in the leather industry. Limitation of evidence: the study was used the volume of microliter (μL) which is a minimal amount and did not show how it was used when reacting with 0.08U HRP. Furthermore, the study did not provide enough information about the free HRP enzyme, resulting in an unanswered research question.

Evaluation The study of (Sadhanandam et al, 2013) was chosen in an attempt to ensure consistency of result about whether a free HRP or immobilised HRP enzyme is more efficient in terms of contact time and H2O2 concentration. It is reliable source that was published in a well-known journal (A SpringerOpen Journal) in 2013. This study was focused on the advantage of immobilised HRP enzyme than free HRP enzyme.

Suggestions of improvements -

The unit of the H2O2 need to be convert to the real-life unit. More detailed about the H2O2 concentration react with HRP enzyme to determine weather the free HRP or immobilised HRP is more effective.

Suggestions of extensions -

The recyclability of the free HRP enzyme in order to compare stability of the immobilised HRP and free HRP enzyme.

Conclusion The study was used to determine whether immobilised HRP enzyme is more effective than the free HRP enzyme have not led to any conclusive results. Although the free HRP enzyme has a faster reaction time, the immobilised HRP enzyme can be recycled and give it an advantage in industrial applications. While the study review does not answer the research question, it does support the claim that catalysts and enzymes are the manufacturing industry's future.

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