Standard using scanning electron microscopy (SEM), atomic force

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Last updated: September 28, 2019

Standard testingmethodsVisualobservationsVisualobservations include roughening of the surface, formation of holes or cracks,de-fragmentation, changes in colour, or formation of bio-films on the surface.To study degradation mechanism, observations can be made using scanningelectron microscopy (SEM), atomic force microscopy (AFM) (Ikada, 1999), Fouriertransform infrared spectroscopy (FTIR), differential scanning colorimetry(DSC), nuclear magnetic resonance spectroscopy (NMR), X-ray photoelectronspectroscopy (XPS), X-ray Diffraction (XRD), contact angle measurements andwater uptake.  Weight lossmeasurements: determination of residual polymerWeightloss measurement is widely applied in degradation tests but no direct proof ofdegradation is obtained. Procedure like DIN V 54900 and sieving analysis of thematrix surrounding the plastic sample allows quantitative determination of thedisintegration characteristics.

For polymers like powders the decrease inresidual polymer can be determined by an adequate separation or extractiontechnique (polymer separated from biomass, or polymer extracted from soil orcompost). By combining a structural analysis of the residual material and thelow molecular weight intermediates, detailed information regarding thedegradation process can be obtained, especially when a defined synthetic testmedium is used (Witt et al., 2001). Changes in mechanicalproperties and molar massMechanicalproperties are an important parameter to determine the minor changes in themass of the test specimen. Properties like tensile strength which is verysensitive to molar mass change, can be directly taken as an indicator ofdegradation (Erlandsson et al.,1997) (Atefeh et al.

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, 2013). For enzyme induceddepolymerisation  the material propertiesonly change if a significant loss of mass is observed (the specimen becomethinner because of the surface erosion process; the inner part of the materialis not affected by the degradation process), for abiotic degradation processes(which often take place in the entire material and include the hydrolysis ofpolyesters or oxidation of polyethylenes) the mechanical properties may changesignificantly, though almost no loss of mass due to solubilization ofdegradation intermediates occur at this stage. As a consequence, this type ofmeasurement is often used for materials where abiotic processes are responsiblefor the first degradation step (Breslin, 1993; Tsuji and Suzuyoshi, 2002) (Shahet al.

, 2007).Themelting point is another parameter and it was found that the melting point offungal treated polythene strips has reduced to 161ºC when compared to control162.2ºC polythene strips (Raman et al.

, 2012). Degradation has been reflectedthrough changes of material properties such as mechanical, optical, electricalcharacteristics, crazing, cracking, erosion and discoloration phase separation.The change includes bond scission, chemical transformation and formation of newfunctional groups (Sharma et al.

, 2013)  CO2 evolution/O2consumptionUnderaerobic conditions, microorganisms use oxygen to oxidize carbon and form carbondioxide as one of the major metabolic end product. So the consumption of oxygen(Respirometric test) (Hoffmann et al., 1997) or the formation of carbon dioxide(Sturm test) are good indicators for polymer degradation, and are the mostoften used methods to measure biodegradation in laboratory. Besidesconventional trapping of CO2 in Ba(OH)2 solution,followed by manual titration, infrared and paramagnetic O2 detectorscan also be used to monitor O2 and CO2 concentrations inthe air stream. In order to detect minute concentrations of CO2 or O2methods like trapping CO2 in a basic solution, with continuoustitration or detection of the dissolved inorganic carbon (Pagga et al., 2001)may be useful. For non-continuously aerated, closed systems CO2 canbe detected by a sampling technique in combination with an infrared-gasanalyzer (Calmon et al.

, 2000) or a titration system (Mueller, 1999). Therespiratory activity was measured by coupling the flasks to a continuous-flowrespirometer coupled to an infrared CO2 detector (TR-RM8Respirometer Multiplexer–Sable systems) (Albino Paes et al., 2015). Anotherdiscontinuous titration method for closed system has been described by (Solaroet al., 1998), using small closed bottles as degradation reactors and analyzingthe CO2 in the headspace (Itavaara and Vikman,1995) or by thedecrease in dissolved oxygen (closed-bottle test) (Richterich et al., 1998) aresimple methods. RadiolabelingNet CO2and 14CO2 evolution measurements are simple, non-destructiveand measure ultimate biodegradation. Materials containing a randomlydistributed 14C marker can be exposed to selected microbial environments.

Theamount of 14C carbon dioxide evolved is estimated using a scintillationcounter. This method shows a high degree of precision and consistency (Sharabiand Bartha, 1993). The drawback with this method is that the labelled materialsare costly, the licensing and waste disposal problems connected withradioactive materials.  Clear-zoneformationVery simpleagar plate test in which the polymer is dispersed as very fine particles withinthe semi synthetic or synthetic medium, after inoculation with the testorganism; the formation of a clear hole around the colony indicate that thetest organism is able to depolymerise the polymer. This method is usuallyapplied to screen organisms that can degrade a certain polymer (Nishida andTokiwa, 1993; Abou-Zeid, 2001), but it can also be used to obtainsemi-quantitative results by analyzing the growth of clear zones (Augusta etal., 1993). Viabilitytest Viabilitytest by pour plate method to examine the living existence of the microbes helpin obtaining the information that micro-organisms are acquiring energy bybio-degradation of polymers available in their surrounding (Dey et al., 2012).

 EnzymaticdegradationThedegradation of polymer by microorganisms involve two steps ;  first they bind to the polymer surface andthen catalyses hydrolytic cleavage. The polymers can be degraded by the actionof intracellular or extracellular enzymes. Intracellular degradation is thehydrolysis of an endogenous carbon reservoir by the accumulating bacteriathemselves while extracellular degradation is the utilization of an exogenouscarbon source not necessarily by the accumulating microorganisms (Tokiwa andCalabia 2004). During degradation, extracellularenzymes from microorganisms break down complex polymers yielding oligomers,dimers, and monomers that are smaller enough to pass the semi-permeable outerbacterial membranes. These short chain length molecules are then mineralizedinto end products e.g.

CO2, H2O, or CH4, thedegradation is called mineralization, which are utilized as carbon and energysource (Frazer, 1994; Hamilton et al., 1995). (Gu et al., 2003).  Evaluation ofbacterial hydrophobicityBacterialcell-surface hydrophobicity was estimated by the bacterial adhesion tohydrocarbon (BATH) test (Rosenberg et al. 1980), which is based on the affinityof bacterial cells for an organic hydrocarbon such as hexadecane.

The morehydrophobic the bacterial cells, the greater their affinity for thehydrocarbon, resulting in transfer of cells from the aqueous suspension to theorganic phase and a consequent reduction in the turbidity of the culture. (Hadadet al.,2004).


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