Biological degradation of natural rubber glove by gram negative bacteria Klebsiella aerogenes

As the urgency and the scope of the global threat posed by the rubber waste is increasing, so have the efforts to �nd sustainable and e�cient solutions. In recent years, biological degradation of rubber waste has been gaining interest as an alternative to conventional waste management practices and widely used chemical methods. The gram-negative bacteria Klebsiella aerogenes was used in the current study to perform natural glove rubber (NGR) degradation. Parameters such as fermentation duration (within 21 days), temperature (30, 35 and 40 o C) and pH (6,7 and 8) on the effect of biodegradation was investigated. The biodegraded rubber was characterised by dry weight analysis which showed a maximum weight loss of 17% at pH 7 under 35 o C after 21 days. The growth curve analysis showed that a maximum cell concentration of 4.12 g/L in week 2 of the biodegradation process. Increase in viable cell count over the incubation time indicated that rubber waste had suitable carbon source to sustain the culture during the biodegradation process. The visual con�rmation of biodegradation was carried out by Schiff’s staining indicating the formation of aldehydes and ketones. Further con�rmation on the presence of new aldehydes and ketones peaks were shown in FTIR analysis. Results also showed that highest protein concentration of 657.02 µg/ml and enzyme (laccase and Manganese peroxidase) activity of 0.43 ± 0.05 IU was observed at week 2 of the biodegradation. The experiment results concluded that Klebsiella aerogenes had the highest rubber weight loss in shorter period. This paper is �rst to report the presence of laccase and MnP enzymes in Klebsiella aerogenes. The bacteria Klebsiella aerogenes is e�cient in degrading rubber in a shorter period, further analysis on the biodegradation pathway (mechanism) is worth investigating.


Introduction
Rubber is a hydrocarbon polymer that is used to make a variety of consumer, commercial, and industrial goods.Only 1.7 million tonnes of the 9.2 million tonnes of rubber trash generated were determined to be recyclable.Taking land lls and energy recovery out of the equation, this statistic represented only 40% of all tire trash produced [1,2].In 2018, 7.2% of rubber waste was burned for energy recovery, and 3.4% was disposed of in land lls.In addition to tires and home appliances, 30% of the rubber market is made up of consumer products like shoes, toys, sporting goods, and leisure items as well as general rubber goods like belting, hoses, and tubes.Unfortunately, there is currently relatively little effort put into recycling the waste produced by the general rubber goods business.There is merely 1.5% of the waste produced is recycled or reused from typical rubber products [2,3].
The challenge of waste rubber disposal is one of the major concerns worldwide.Conventional waste management practices like burning and open dumping of the rubber waste causing major pollution issues [2].The leading cause of the ecological problems caused by the rubber waste are inadequately established technologies and improper waste management procedures.Focus on sustainability is necessary for rubber industry to tackle the inherent challenges posed by different kind of rubber waste produced [2].Currently thermal, mechanical, and chemical methods are heavily documented.In recent times most efforts are directed towards biological method due to the environmentally friendly nature [4,5].
Degradation of natural rubber through biological methods was rst studied in 1914 by using solution cast lms of natural rubber as a carbon source [2,6].Biological degradation of rubber is a slow process with incubation periods extending over weeks, months, or years to see signi cant degradation.According to several studies the actinomycetes are the major bacteria capable of degrading the rubber in considerable amounts.Two distinct kinds of rubber degrading bacteria are reported, the ones which form clear zones and the ones which degrade by adhesive action [7].Nocardia sp 835A and Nocardia sp.835A strains have been reported to degrade about > 90% and 80% of the rubber material by adhesive action with minimum incubation period of 8 weeks.Various bacteria belonging to the actinomyces such as Streptomyces coelicolor CH13, Streptomyces sp.K30, Corynebacterium, Nocardia, Gordonia sp.Mycobacterium group, Alicyclobacillus sp. were identi ed as rubber biodegrading bacteria that produce a clear zone [2,7,8].
Clear zone formation is by producing enzymes by the bacteria that aid in the biodegradation process.
Rubber oxygenase A (roxA), rubber oxygenase B (roxB), latex clearing protein (lcp), laccase, and manganese peroxidase are the major enzymes that have been discovered till date that degrade rubber [9].Lcp has been considered a key enzyme in biodegradation of natural rubber that was reported in gram positive bacteria like Streptomyces sp.Whereas, roxA is reported in gram negative species like Xanthomonas strain 35Y [9,10].Laccase and manganese peroxide (MnP) are commonly produced by white rot fungi but are also reported in bacteria.Bacillus pumilus, Bacillus subtilis, Paenibacillus sp., are reported to produce laccase and MnP that catalyse the biodegradation process [11,12].Bacillus sp.SBS could grow under a temperature range of 15 to 42 o C and a pH range of 5 to 10 with maximum degradation of 2.03% weight loss was reported after a 5 day incubation at 30-35 o C at pH 7 [13][14][15].Majority of the reported rubber biodegrading bacteria were gram-positive.Xanthomonas sp. is the only gram-negative bacteria that have been reported till date.The bacteria were capable of degrading rubber by producing roxA enzyme when incubated for 10 weeks at 30 o C. Maximum weight loss of 60% was reported at the end of the incubated period [16].The mechanism of roxA enzyme has been studied extensively whereas mechanisms of laccase and MnP have not been reported yet [16].Until now laccase and MnP have been only reported in gram-positive bacteria for biodegradation, but none have been reported in gram-negative bacteria for rubber degradation.The ability of the gram-negative bacteria to produce a wide range of extracellular enzymes like laccase and manganese peroxidase besides RoxA, RoxB and Lcp makes them advantageous over gram-positive bacteria [17] They also possess a unique lipopolysaccharides layer which is absent in gram-positive bacteria making them resistant over environmental stresses such as pH and temperature uctuations occurring during the biodegradation [18].The layer also gives protection from toxic secondary metabolites formed during the incubation period.Furthermore, gram-negative bacteria have higher metabolic enabling them to consume wider range of compounds as carbon source [18].Studies have also shown that gram-negative bacteria show 60% of rubber degradation rate in shorter periods of time compared to gram-positive bacteria [2].
In the current paper the rubber biodegrading ability of gram-negative bacteria Klebsiella aerogenes was investigated.The bacteria Klebsiella aerogenes has been shown to have high e ciency in degrading polymers like polyethylene terephthalate (PET) and polystyrene (PS) which are commonly used plastics that are di cult to degrade [19,20].It is known to produce extracellular enzymes like peroxidases which aid in polymer biodegradation [19,20].Klebsiella aerogenes is abundantly available in the soil, less toxic and able to utilize a variety of carbon sources for growth.In this study, submerged fermentation was proposed for bacteria cultivation which is advantageous than solid state fermentation in terms of diffusion of nutrients, measuring, and controlling of operating parameters [21].
In this study, the optimum conditions for the growth, enzyme production and the characterization of both enzymes and the biodegraded rubber has been reported.

NGR sample preparation
Natural glove rubber (NGR) was cut into small pieces ranging from 1cm to 4cm.The NGR glove pieces were soaked in 90% ethanol for 24 hrs to remove any external impurities.After ltration, NGR were washed thoroughly in water and dried in an oven at 50 o C until constant weight is reached.

Cultivation of microorganisms
Bacteria Klebsiella aerogenes ATCC No.13048 was used in this experiment The medium used to culture the bacteria was as follows: yeast extract 2g/L, peptone 5g/L, NaCl 5g/L, agar 15g/L was autoclaved at 121 o C for 15 min.The bacteria were cultured at pH 7 and incubated 37 o C for 21 days.

Biodegradation of NGR by Klebsiella aerogenes
1g of NGR pieces and 0.1 ml of bacterial inoculum with 9 x10 6 CFU/ml concentration were introduced in 150ml of the culture media.The pH of the culture media was adjusted to 6, 7 and 8.The culture was incubated in a rotary shaker incubator (YIHDER LM-400D) at 150 rpm for 21 days.The operating conditions investigated are three different pH (6,7 and 8) and temperatures (30,35 and 40 o C) for 21 days.Experiments were conducted in duplicates.The untreated NGR without inoculum was taken as control.The concentration of bacteria was observed every day for 21 days.After 21 days the NGR pieces were ltered and washed with water.The washed NGR pieces are then dried in an oven at 50 o C until constant weight is achieved.Analytical methods

Cell concentration determination
The concentration of bacteria was determined by measuring the absorbance of the samples at 600 nm using UV-Vis spectrophotometer ( Genesys 10S).The concentration of the bacteria was determined by preparing the cell cultures of known concentrations and counting them by using a haemocytometer.The absorbance of the culture was taken at 600nm by using UV-Vis spectrophotometer (Genesys 10S).The standard curve was obtained by plotting the absorbance values against the cell concentration.

Dry weight analysis before and after biodegradation
After 21 days of incubation, the NGR was collected by ltration from the culture asks.The rubber pieces are then soaked in 99% ethanol for 24 hrs.The rubber pieces were then washed thoroughly with water and dried at 50 o C until constant weight is achieved.The weight loss of the rubber was calculated based on the dry weight of the NRG pieces before (W 1 , g) and after incubation (W 2 , g).The changes in the weight were measured by using the formula shown in (1) whereby W 1 and W 2 were determined using an analytical balance with a readability of 0.1 mg.

Viable cell count
The viability of the bacterial cells at the end of incubation period was determined by viable cell count test by spread plating.0.1ml of the diluted bacterial culture by 6 times was spread evenly over the nutrient agar surface.The cell count of bacteria was determined after 72 hr.The viability of the cells after biodegradation process is calculated by using ( 2). (2)

Schiff's staining analysis of the biodegraded rubber
Schiff's reagent is used to detect the aldehyde glycogen and polysaccharide in the NGR and thereby to con rm degradation of hydrocarbon chain [22].The biodegraded NGR at the end of incubation was stained with 5ml ml of fuchsin reagent at room temperature for 45 mins.The Fuchsin reagent consists of 1% of basic fuchsin hydrochloride, 1% of hydrochloric acid, 1% of sodium metabisul te and 98% water.
After 45 mins, colour change was observed as the presence of aldehyde group will change the samples to dark pink.

FTIR analysis of the biodegraded NGR
Functional group changes that occur in the NGR surface due to the biodegradation were determined by using FTIR (PerkinElmer spectrum 100) which has a transmission range of 4000 to 400 cm − 1 .ATR analysis method with 16 scans was used in this experiment.

Protein estimation by Bradford assay procedure
The protein concentration was determined by Bradford assay using Bovine Serum Albumin as a standard.5µg/ml to 25µg/ml of BSA is used in estimating the amount of protein.50 µl of the sample or standard was added with 1.5 mL of Bradford reagent and incubated at 30 o C for 15 min.The protein concentration was determined by absorbance at 595nm using UV-VIS spectrophotometer.

Determination of laccase and manganese peroxidase enzyme activity
The substrate used to measure the enzyme activity is 2mM guaiacol in 10mM sodium acetate buffer (pH 5).The reaction mixture contained 3ml of 10 mM acetate buffer of pH 5, 1 ml of guaiacol and 1 ml of enzyme source. 1 ml of distilled water instead of enzyme source is taken as enzyme blank.The mixture was incubated at 30 o C for 15 min in a water bath and the absorbance was recorded at 450nm.For manganese peroxidase, the activity was calculated by following the laccase enzyme activity procedure but with 1ml of H 2 O 2 added to the reaction mixture and incubated.The temperature and incubation period are the same as laccase assay.Laccase and manganese peroxidase activity was calculated by using the formula(3).
Enzyme activity (U/ml)= The effect of pH and temperature on biodegradation of NGR was investigated in this study.After biodegradation, the nal pH of all samples falls into the range of 9 to 9.5.This might be due to secondary metabolites accumulation like carbon dioxide and hydrogen gas.Although smaller amounts of acids such as lactic acid, acetic acid and formic acid are formed during the fermentation process larger amounts of ethanol are produced throughout the fermentation course resulting in higher pH regardless of the initial pH of the medium [23,24].
As seen from Under all temperatures investigated, pH 7 was found to report the maximum percentage of degradation whereas the minimum percentage of degradation was recorded in samples with initial pH of 8. Similar ndings were reported where pH 7 and 37 o C were studied as the optimum conditions for the biodegradation process for Klebsiella species [26,27].This is because temperature affects bacterial growth, metabolism, rate of biodegradation and enzymes production [26,27].The pH of the surrounding environment greatly in uences the cell structural integrity and cell metabolism.pH also has an impact on kinetics and thermodynamics of microbial respiration in uencing the structure and function of the bacteria [26,27].pH affects the bacteria directly by affecting the consumption and production of protons whereas indirect affect is caused from regulation of chemical speciation [28].
In a study by Nayanashree et al. and A.A. Shah et al. [11,29] maximum weight loss of 29.3% was reported at the end of 6 months when bacterium Bacillus subtilis [11] and Bacillus pumilus [29] were applied.The optimum temperature for the bacteria Bacillus subtilis [11] and Bacillus pumilus [29]was reported to be 37 ± 2 o C. Another study on natural rubber biodegradation by soil microorganism was conducted for a period of 236 days resulted in a dry weight loss of 15.6% [4].On the other hand, studies on biodegradation of NR by using fungus reported a weight loss of 4.3% over an incubation period of 65 days when Alternaria alternata was used [30].Fungus P.chrysogenum showed a weight loss of 28.3% after an incubation period of 6 months.Weight loss of 18.82% was reported from mixed culture of Rhodococcus pyridinivorans after 1 month of incubation [31].From the above reported studies, it is evident that Klebsiella aerogenes is proven advantageous in biodegrading NGR in shorter time periods in this study.

The effect of temperature and pH on the growth of bacteria
The growth of the bacteria under different conditions of pH (6,7 and 8) and temperatures (30,35  This indicated that 35 o C and pH 7 might be the most effective conditions to promote cell growth and degradation of rubber.these results can be aligned with the results of dry weight analysis in which maximum degradation of 17% was observed at the same parameters.
After the end of the incubation period, cell viability analysis was conducted to assess whether the bacteria are still alive after the degradation of NGR.It was found that the cells were viable at the end of incubation indicating that the nutrients provided were su cient to sustain their growth.In the present study, higher cell viability was found in pH 7 at 30 o C, 35 o C and 40 o C as seen from Table 2.The bacteria showed higher cell count after end of the incubation period at all the temperatures and pH than at the beginning of the biodegradation process con rming that bacteria have consumed the NGR pieces for its growth.Although, there are differences present in viability of the cells present in solutions of pH 6, 7 and 8 with at 30 o C, 35 o C and 40 o C, they are insigni cant.The decrease in the bacterial viability at 40 o C might be due to the increase in temperature.The bacterial yield is affected by higher temperatures.Though the temperature is suitable for growth is might not be suitable for product formation which can been seen at all pH at 40 o C. In the biodegradation process higher temperatures acted as limiting factor resulting in lower cell growth and lower product yield.The higher the temperature the lower the bacterial viability due to cell rupture and cell death.5.3x10 7 ± 2.9x10 5   3.2x10 7 ± 1.3x10 5   5.1x10 7 ± 1.4x10 5   5.6x10 7 ± 2.4x10 5   4.3x10 7 ± 1.2x10 5   4.6x10 7 ± 2.4x10 5   5.1x10 7 ± 1.4x10 5   4.1x10 7 ± 1.8x10 5 7 8

Schiff's staining analysis of biodegraded rubber
To further con rm degradation of rubber, Schiff's analysis was performed to check the presence of aldehydes and ketones groups due to degradation of cis-1,4 polyisoprene in the natural rubber by the bacteria.Figure 2 clearly shows that aldehydes and ketones group is present in a signi cant amount when NGR samples show deep pink colour after Schiff's staining.The intensity of the colour obtained indicates strong presence of aldehydes in the biodegraded rubber which was a result of biodegradation [11,12,29].The biodegraded rubber produced similar results when treated with Bacillus subtilis, Bacillus pumilus, Bacillus sp.AF-666 l [11,12,14,15,29,34,35].Formation of aldehydes and ketone groups by the action of roxA enzyme was also reported in the gram-negative bacteria Xanthomas sp.35Y strain [9,10,36,37].The control NGR pieces showed no colour change indicating no biodegradation occurred.

Analysis of the functional groups
Signi cant biodegradation of NGR by Klebsiella aerogenes after a period of 21 days was evident from dry weight and Schiff's staining analysis.In Figs.3,4 and 5 when the control sample was compared to that obtained from biodegradation, there were signi cant differences in the peaks obtained.Spectrum of the control sample showed two peaks that are present at 1576 cm − 1 and 1539 cm − 1 that correspond to C = C bonds in the NGR.In the biodegraded NGR spectrum of pH 6,7 and 8 samples, C = C peaks disappeared and new peak at 1663 cm − 1 that correspond to aldehydes and ketones(C = O) were formed at all investigated temperatures [29,[38][39][40][41]. NR degraded by bacteria Paenibacillus lautus showed similar peaks at the band range of 1660 − 1650 cm − 1 indicating aldehydes and ketones formation [41].These peaks corresponding to aldehydes and ketones in the range of 1650-1750 cm − 1 were also present in NR samples inoculated with streptomyces CFMR 7 and Thiobacillus ferroxidans, Bacillus sp.AF-666 and Rhodococcus pyridinivorans strain F5 [31,38,40].Results obtained from FTIR spectrum further supported the observation on Schiff's staining to con rm the presence of aldehydes and ketones.The The biodegradation mechanism of Klebsiella aerogenes on NGR can be described as scission of the polymer chain (C = C) by oxygen attack [31].The attack on the polymer chain resulted in the formation of aldehydes and ketones (C = O).Similarly, oxygen attack on the sulfur bonds in the NGR resulted in the formation of sulfoxides (S = O).As a result, oxidative cleavage might have occurred during the degradation of natural rubber[16, 36, 37].The oxidation reaction might have occurred by utilising the oxygen present extracellularly or by the activation of the oxygen present in the bacterial cell.However, irrespective of the colonizing strategy of the bacteria it is evident from Schiff's staining test that oxidative cleavage is the rst metabolic step in biological natural rubber decomposition [15,29,44].

Trend of total protein concentration during biodegradation
Protein estimation in bacterial cell culture is conducted to study the different kinds of proteins produced by the bacteria during the biodegradation process.The protein production by the bacteria was in uenced by parameters like pH, temperature, and incubation time signi cantly.When incubation time increases, the protein concentration increased in all samples.From Fig. 6b, the maximum concentration of protein was found at 35 o C, pH 7 on week 2 which is 657.02 µg/ml (P > 0.05), compared to 532.31µg/ml (P > 0.05) and 465.1µg/ml (P > 0.05) that was recorded from samples prepared at pH 7 under 30 and 40 o C respectively on week 2. The improvement in protein could be related to the enzyme activity which is crucial for rubber degradation.This indicates that the optimum conditions for enzyme activity was at pH 7 and 35 o C, which coincides with the highest degradation rate and bacteria growth rate.Similar ndings of protein concentration were in the fermentation media of Bacillus pumilus (0.086 µg/ml), Bacillus subtilis, Bacillus sp.S10 (290 µg/ml) and Klebsiella pneumoniae (1.21mg/ml) were reported [12, 15, .

Trend of laccase and Manganese peroxidase (MnP) enzyme activity during biodegradation
The enzymes responsible for rubber degradation includes RoxA, roxB, lcp, laccase and manganese peroxidase as reported previously [2].RoxA and lcp are the most extensively studied enzymes in grampositive bacteria compared to laccase and Mnp.In this study laccase and MnP were both produced in Klebsiella aerogenes in submerged fermentation [47].The effect of temperature and pH on laccase and MnP can be observed in Table 3.There were signi cant differences (P ≤ 0.06) on the enzyme activities reported at different temperature and pH.The optimum laccase activity was found to be at week 2 across all temperatures and pH with 0.43 ± 0.05 IU being the highest at 35 o C.This corresponds to the bacterial cell concentration, growth and protein concentration which were found maximum in week 2.Moreover, there is a signi cant difference (P ≤ 0.05) in enzyme activity recorded at temperatures 30 and 40 o C and pH 6 and 8, which might suggest a reduction in metabolic activities of bacteria.
Similarly, the activity of MnP showed a signi cant difference (P ≤ 0.05) across all temperature, pH, and time.From  [11,12].Form the above results, it is evident that Klebsiella aerogenes produced a higher enzyme activity in shorter duration.This could be translated into biodegradation of rubber could be taking a shorter duration to reach the same weight loss when Klebsiella aerogenes was used.From the ndings of protein concentration and enzyme activity, it is evident that higher protein production is directly related to higher enzyme activity resulting in higher biodegradation rate.[47,48].Till date laccase and MnP that were produced during rubber biodegradation were only reported in Bacillus subtilis and Bacillus pumilus [11,12].Under similar experimental conditions it is seen that Klebsiella aerogenes can produce laccase and MnP that yield higher rubber degradation in shorter periods of time in comparison to bacteria producing RoxA, RoxB. 4 Conclusion In the current study the bacteria, Klebsiella aerogenes was able to degrade the NGR signi cantly in 21 days.The determination of weight loss, increase in the viable cells, Schiff's staining, FTIR analysis has con rmed that biodegradation has taken place.Highest weight loss (17%), higher cell concentration, higher protein concentration and maximum enzyme activity were seen at 35 o C under pH 7. Schiff's analysis of biodegraded NGR con rmed the presence of aldehydes and ketones.The disappearance of 1576 cm − 1 and 1539 cm − 1 peaks in the biodegraded samples during ATR-FTIR analysis con rmed that C = C and S-S cleavage had taken place.Formation of new peaks at 1038 cm − 1 indicated the presence of sulfoxides (S-O) con rming devulcanization.Protein estimation and enzyme activity determination con rmed the production of laccase and MnP.Highest protein production was observed at week 2 which fell in line with highest enzyme activity of both laccase and Mnp at week 2 of biodegradation.Presence of both laccase and MnP in the medium indicated that the NGR underwent oxidation.To summarise, Klebsiella aerogenes have successfully degraded NGR signi cantly in shorter time.The bacteria are also capable of devulcanization in shorter period.Though the basic mechanism of the enzymes could be understood, the exact mechanism is not understood yet and extensive research is lacking.The rubber produced after biodegradation loses its mechanical properties because of carbon chain cleavage making it not useful for further reclaiming processes.The path of the biodegraded rubber is not clear and further research is necessary.

Figures
Page 18/ Where A = absorbance at 450 nm V = nal volume t = incubation time e = extinction coe cient (0.6740 µM/cm) V s = enzyme volume 3 Results and Discussion 3.1 Effect of temperature and pH on biodegradation of NGR and 40 o C) has been investigated.From Fig.1, it can be observed that the growth curve at all the temperatures and pH investigated was exhibiting very distinct lag, exponential and stationary phase.Similar growth curve trend was seen with Klebsiella sp.grown in LB medium at 30 o C, 37 o C and 40 o C[32,33].In the current study maximum cell concentration was found to be at 35 o C for pH 7 In pH 7 at 35 o C highest bacteria concentration of 0.68 g/L (Fig.1b) was found on day 11 of the biodegradation from which the stationary phase can be observed.The cell concentration in the stationary remains constant for some period as seen in Fig.1(a),(b) and (c) due to active cell metabolism.During this period the bacteria produces secondary metabolites which result in decline in growth The maximum cell concentration of 0.52 g/L and 0.59 g/L were obtained at 30 o C and 40 o C at pH 7. The cell concentrations recorded at the remaining parameters were lesser than 0.68 g/L obtained at 35 o C.
wavelength corresponding to 1012 cm − 1 in the control NGR indicated the presence of carbon backbone (C = C).However, in the biodegraded NGR samples, the peak shifted to the formation of a new peak at 1084cm − 1 is seen which indicates formation of alcohols (C-O) which is similar to the ndings of NR degraded by Bacillus subtilis, Pseudomonas aeruginosa and Streptomyces sp., Bacillus sp.AF-666[13,29,35,42].Interesting, the cleavage of sulfur bond (S-S) was observed by Klebsiella aerogenes on the formation of new peak at 1038 cm − 1 indicates the presence of sulfoxides (S = O).Disappearance of C = C and S-S groups and formation of aldehydes, ketones, alcohols and sulfoxide groups prove that cleavage of the NGR backbone had occurred and also sulfur bonds have been broken[43].Similar results were obtained when samples from 35 o C and 40 o C were subjected to FTIR analysis.Cleavage of both C = C and S-S bonds in the NGR indicates degradation and devulcanization by the bacteria[2].

Figure 1 Cell
Figure 1

Table 1
[25] 40 o C might not be suitable for the biodegradation of NGR as the optimum temperature for growth of Klebsiella aerogenes is in the range of 30 and 37 o C[25] weight loss of 15, 17 and 12% were observed when the biodegradation process was conducted at 30 o C under initial pH of 6, 7 and pH 8 respectively.When the temperature increased to 35 o C, an insigni cant difference in weight loss was obtained under different initial pH conditions.However, a lower weight loss of 9, 8 and 10% were recorded at pH 6, 7 and 8 respectively under 40 o C.This implied

Table 2
Bacterial cell concentration at different temperatures and pH

Table 3 ,
the highest MnP activity of 0.26 ± 0.05 IU was seen at week 2 at 35 o C pH 7 which is much lesser when compared to the laccase activity.Also, there is a signi cant difference (P ≤ 0.04) of MnP enzyme activity at temperature 30 o C and 40 o C and pH 6 and 8.The above results indicated that the laccase enzyme activity was higher when compared to MnP activity.After 4 months of biodegradation, 0.03 IU of Mnp activity was reported in A.alternata. 0.0105 IU and 0.012 IU of laccase and MnP activity was reported in Bacillus sp., after 8 weeks of incubation respectively

Table 3
Laccase and Mnp activity at different duration, temperature, and pH Production of laccase and MnP by bacteria could give an insight into the mechanism of enzymes in the biodegradation process.Both enzymes are oxidoreductases that catalyse the reaction by free radical mechanism turning substrate into oxidised or polymerised products.Laccases catalyse the reaction by using oxygen as an electron acceptor whereas MnP oxidises through hydrogen peroxide dependent oxidation.The presence of both the enzymes indicated that NGR possibly underwent oxidation in biodegradation process.The general mechanism of MnP begins when the heme group transfers two electrons to H 2 O 2 in its resting state producing water and compound I.The second step involves oxidation of Mn 2+ by compound I forming compound II, Mn 3+ and a free radical.The formed compound II catalyses Mn 2+ to Mn 3+ and MnP reverts to its original form.The mechanism can be summarised as follows:Laccases are copper containing enzymes which degrade substrates like aromatic amines, amino phenols, aliphatic amines by oxidising them using oxygen and forming water.The above depicted mechanisms are the catalytic biochemical reactions of both laccases and MnP in the presence of the substrate.Although it is understood that the enzymes degrade NGR by oxidation the exact mechanism of both the enzymes is still not known.Different types of rubber degrading enzymes by different bacteria are given in the Table4.The most studied enzymes RoxA, RoxB are found in gram-negative bacteria while Lcp is most found in grampositive bacteria.Although laccases and MnPs have several applications in textile waste their use in rubber biodegradation is relatively new

Table 4
Different kinds of biodegrading enzymes produced by different bacteria and weight loss obtained in the process.