PLA biodegradation

1. DEGRADATION OF POLYLACTIC
ACID AT MESOPHILIC AND
THERMOPHILIC CONDITIONS
SEMINAR 1(1-0)
GROUP MEMBERS:
 AIMA KHALID 16-ARID-2533
 FARYAL AKHTAR 16-ARID-2547
 SABAHAT ALI 16-ARID-2569

2. CONTENTS:
 INTRODUCTION OF PLA
 TEMPERATUREINFLUENCE ON REACTION RATE BY
ARRHENIUS EQUATION
 EFFECT OF MESOPHILIC ALKALINE BACTERIUM ON PLA
 EFFECT OF MESOPHILIC TEMPERATUREON n PLA and c
PLA DEGRADATION:
 MESOPHILIC BACTERIA ISOLATED FROM COMPOST
DEGRADE POLYLACTIDE
a. RECOVERY OF PLA FILM
b. SCREENING VALIDATION
 CRITICAL EVALUATION
 CONCLUSION/SUGGESTION
 ANAEROBIC BIODEGRADATION TEST OF POLYLACTIC
ACID UNDER THERMOPHILIC CONDITIONS
 ESTIMATION OF THERMAL DEGRADATION OF PLA BY
THERMOGRAVIMETRIC ANALYSIS
 DEGRADATION OF POLYLACTIC ACID BY PLA
DEPOLYMERASE ISOLATED FROM THERMOPHILIC
BACILLUS
 ISOLATION OF THERMOPHILES DEGRADING POLY LACTIC
ACID
 THERMOPHILIC ANAEROBIC BIODEGRADATION OF PLA
 CRITICAL ANALYSIS
 SUGGESTION

3. POLYLACTIC ACID:
Polylactic acid is an excellent bioplasticto be known because
of its excellent biodegradabilityas well as its wide range of
application.
 Mesophilicbacteria usuallygrows at a moderate
temperature and shows optimum activity at 68 to
113F[20 to 45C]while thermophiles usually thrives at a
very high temperature 106 to 252F[41 to 122]
 Mesophilicconditionare mostly conducive for activity
and microbial growth.
 During winter or at low temperature the number and
microbialactivity decreases(10 to 15 degree celsius)

4.  Temperature influenceon reaction rate is described by
Arrheniusequation:

5. At three different soil temperature and retrieval time the
table provides Mn and PDI value for MB non -woven mulches.
At 23 degree A large decrease of Mn for MB PLA +PHA-
75/25,27 percent was measured at 10 week
As compared to 20 and 30 week this decrease was much
larger as compared to 10 week

6. At temperature T1 and T2 the reaction rate are R1 and R2 ,
Q10 is temperature coefficient.
 According to equationif the soil temperature increases
by 10K at 308.15K the biodegradation rateconstant of
PLA increase 1.5 fold.
 Therefore by the minor increase of temperature the
biodegradation rateof PLA based mulches increases
substantially.
 In order to understandthe biodegradationof MB
mulches in a warm environment an effort was made to
stimulate the elevated soil temperature.
 During moderate weather[autumn and spring]the soil
temperature was stimulated by employing laboratory
ambient conditionand it yield lower air temperatures
than greenhouse condition.

7.  Three different soil temperature employedin this. in the
greenhouse environment ambientsoil
temperature[control], above ambient soil temperature at
3 degree Celsius by employingthe heat method
 By employing ambient soil conditionin the laboratoryin
the green house at 5 degree Celsius compared to
ambient soil temperature in green houses.

8. After 10 weeks increase of temperature decreases the tensile
strength of MB non- woven mulches significantly.
 At the soil temperature of 23 degree the tensile strength
loss value was 94 percent for MB PLA +PHA-7525.This
loss is attributableto microbialactivity
 At lower soil temperature 15 degree Celsius MB PLA
undergo higher loss of tensile strength value 92 percent
as compared to higher soil temperature[66 percent at 23
degree Celsius]
EFFECT OF MESOPHILIC ALKALINE BACTERIUM ON PLA:

9.  Pseudomonasgeniculata WS3 and s. pavaniiCH1 are
gram negative bacteria and they are of rod-shaped.
These PLA-degrading bacteria can be grown easily in
culture broth with their temperature range from 25 to
40C.The PH well-suited for former is 7.0 to 7.5 while that
of latter one is 8.0 to 8.5. showing that latter one is
mesophilic alkalinetolerantbacterium.
 Here is the PLA film biodegradabilityby S pavanii CH1
and P.geniculata WS3 that was being cultured in BSM
broth having an optimal PH and gelatinconc . the PLA
film-weight loss percentage on bacterialinoculated
sample increased considerablyas compared to the
control uninoculatedone.
 PLA film in culture broth that was inoculatedwith s.
pavanii yielded higher PLA film weight loss as compared
to those inoculatedwith P. geniculata WS3 . The result
proves former better performance on PLA film
biodegradabilitythanlatter.

10. The MW change is supported by the cleaving Of ester bond. In
return, the hydrolytic enzymes cleaving ester linkages are
produced by PLA degrading enzymes.
Castro-Aguime reported that PLA film MW
decreases under mesophilic conditionas compared to
thermophilic.

11. EFFECT OF MESOPHILIC TEMPERATUREON n PLA and c PLA:
While comparing n PLA and c PLA, the biodegradationrate and
percentage of n PLA is higher than c PLA at all treatments.
under the mesophilic conditionunsterilizedsoil mixture that
was cultivated with p geniculataand control one both these
sample has a higher n PLA weight loss than that of c PLA.in
non sterilized soil mixture that was cultivatedwith or without
p geniculate WS3 . The percentage of n PLA and c PLA were
not different. But in the sterilized soil mixture with the
cultivationof WS3 is well suited for n PLA (68.7%) weight loss
than that of c PLA(12.3%).
RESULT:
So the result indicate that WS3 easily degrade n PLA than that
of c PLA. Because the exclusive carbon source used for
screening and cultivating WS3 was a emulsified n PLA.

12. The percentages of n PLA and c PLA inoculated with or
without p geniculata after burial in the sterilized and non-
sterilized mixtures under a] mesophilic b]thermophilic
condition
 In the non-sterilized soil mixture inoculatedwith WS3 ,
under thermophilicconditionat the 12 day of soil burial.
More than 90% of n PLA and c PLA weight loss is
observed while in the 60 days of soil burial under
mesophilic conditionmore than 5-10% of c PLA and n PLA
weight loss is found
 SO here the graph shows that Degradationunder thermal
conditionis higher than at mesophilic condition.

13. The figure is showing the methane ratio during the
biodegradation ofPLA in sludge at 35C and 55C . during the
degradationof PLA the methane ratio that was found to be
stable at 35C was 60% after which the evolutionof gas started

14. in the 55 days. But ratio was low at 55C indicatingthe low
evolved biogasand its stabilitywas found to be b/w 50% and
60% .
During PLA degradation the overallmethane ratio in the total
evolved biogaswas 55% and 56% at 55C.
 Two internationallyrecognized tests are carried out for
evaluatingthe anaerobicbiodegradationof the plastics.
(ISO 15985, 14853) and American Society for Testing and
Material(ASTM) and (ASTM D5526, D5210, etc).
 A mixed microbial population derived from compost
and waste water treatment facility was cultivatedin
a synthetic growth medium. ASTM5210 and an
equivalentISO14853 are aquaticbiodegradationtest
at mesophilictemperature are performed at these
microbial population.
 The basic principles of these tests is dilutingthe
sludge with medium containingthe mineral salts.
The results is only those microorganisms which
primarily use the polymer as carbon source will be
grown . This populationwill not reflect the original
sludge. ASTM D5526 uses 20% TS a high-solid
concentrationwith mixed inoculum
 anaerobicdigester that was only operated at
pretreated
householdwaste is used to derived mixed inoculum.

15. DEGRADATION OF POLYLACTIDE BY MESOPHILIC
BACTERIA DERIVED FROM COMPOST:
Geo bacillusthermocatenulatus, Bacillussmithiiand
Brevibacillusare being observed to exhibit PLA degrading
activity.
There are some bacteria like PaenibacillusAmylolyticus,
Bordetella petrii and Pseudomonas are found to be
mesophilic because they are grown at mesophilic
temperature.
 To determine the decrease in molecularweight of
PLA, the biodegradation ofpolymer can be easily
measured. For this purpose 5 isolatesare taken
named as MS1 ,MS2,MS3 ,MS4 and MS5.

16.  Considerabledifference in PLA film before [day
1]And after [25 days]degradationwas found. The
percentage of weight loss of PLA vary from 10 to
45.5 . this variationshow the effect of compost
isolated on the PLA degradation.The high
percentage of weight loss as shown by isolatesare
MS1[25%] MS2[45.5%] and MS3[27.8%]
 The weight loss is due to microbial activity.
RECOVERY OF PLA FILM:
 In order to assess the nature of microbialgrowth
and surface damage due to PLA film PLA film was
recovered from culture broth
 In control without inoculationof strain the PLA film
was recovered
 Large number of pit holes was seen over the surface
of PLA film incubatedwith pit holes and the film was
attacked by bacillusbacterium

17.  Similarpattern was observe with isolate with MS1
and MS3 respectively.
 VALIDATION OF SCREENING:
For screening proof, the first three isolates MS1 MS2 and
MS3 were inoculatedon emulsified agar plate and zone
of clearance is redetected. The first three isolates
hydrolyze/degrade the PLA film at the mesophilic
temperature of 37C after 20 days of incubation.the
diameter of MS1 MS2 and MS3 was found to be 10mm
28mm and 8mm.which clearly show the high degradation
potentialof MS2 as compared to MS1 and MS2.
Morphological,biochemical andphysiologicaltest shows
MS2 was bacillusstrain which is a mesophile.
CRITICAL EVALUATION:
When it comes to greenhouse and lab conditionthat was
well equippedwith mesophilic temperature then it is
seen that at 20 and 30 week of retrieval PLA film weight
losses much more as compared to 10 week. Well the rate
constant increases much more when the temperature
increases by 10K it increase to 1.5 fold. Laboratory
ambient conditionyield lower temperature than green
hose condition.
SUGGESTION:
It concludes that here degradationof PLA is very much
observant from the decrease in molecularweight at
times degradationis more at mesophilicbut mostly the

18. result shows that for the best degradationof PLA
thermophilictemperature is comparativelymore
favorable than mesophilicbecause PLA degrading
enzymes works best at high temperature.
DEGRADATION OF POLYLACTIC
ACID AT THERMOPHILIC
TEMPERATURE

19. POLYLACTIC ACID DEGRADATION:
 PLA is a thermoplastic polymer. It has lots of applications
in everyday life. It is a biodegradableand compostable
polymer and can be degraded by different
microorganisms and enzymes.
 However, temperature also affects its biodegradability.It
shows greater degradationat thermophilictemperature
i.e. 55Ç and lesser degradationat mesophilic
temperature i.e. 35Ç.
 Its degradationis very necessary to reduce
environmentalpollution.PLA can be converted to lactic
acid by a process called hydrolysis.
 Its degradationrate may vary from weeks to months
dependingupon the conditions.
ANAEROBIC DEGRADATION TEST OF POLYLACTIC
ACID UNDER THERMOPHILIC CONDITIONS
 It is the method in which plastic determinationof
anaerobicbiodegradationanddisintegrationoccur under
high solids anaerobicconditions.
 It is a method by analysisof released biogas. MODA-B is
used to measure biodegradabilityof plastics. In this
system, during the first 5-6 days, the biogasevolution
was at low levels.

20.  The evolvedbiogas ratio increased after 6 days. However,
the methane ratio in the total PLA degradation is 55%
and 56% at 55Ç. Thismethod is considered to be very
convenientand effective for the anaerobic
biodegradation ofbioplastics.
 The evolvedgas is collected in a gas sampling bag at
atmospheric temperature, where its evolutionis directly
measured.
 With this system about 90% of PLA degradationoccur at
thermophilictemperature. Total solid concentration of
used sludge were 2.07% at 55Ç. PLA was degraded about
60% in 30 days, 80% in 40 days and 90% in 60 daysat 55Ç
which is very effective and time saving as compared to
other methods.
 The table below shows PLA degradationat two different
temperatures.

21. ESTIMATION OF THERMAL DEGRADATION OF PLA BY
THERMOGRAVIMETRIC ANALYSIS
 In this method it is estimated that thermal degradationof
PLA is a one stage process in which the weight loss is 0-
30% and activationenergy becomes 21-23 kJ/mol.
 During the early stage of thermal degradationof PLA the
carbonyl-carbon,oxygen bond cleavage is more efficient
than other as it reduces the number of ester linkages and
increases the amount of carbonylend groups.
 Thermogravimetric analysisis used by using the
isothermal and dynamic modes. The small value of
activationenergy shows that PLA is highly sensitive to
thermal degradation.
 The more general Kissinger method was used to measure
the kinetic constant using eq:
In(r/Tm 2) = ln [(n·R·A·Wm n-1)/E]-E/ (RTm)
where r is the heating rate in the TGA experiment, Tm is the
temperature at the maximum rate of weight reduction, R is
the universal gas constant, E is the activationenergy, A is the
pre-exponentialfactor, Wm is the weight of the sample at the

22. maximum rate of weight loss, and n is the apparentorder of
the reaction with respect to the sample weight.
 The value of E can be estimated from a plot of ln(r/Tm 2)
versus 1/Tm for the variousheating rates. The only one
peak in the plot shows that PLA degradation is a one
stage process associating with the cleavage of ester
bonds.

23. DEGRADATION OF POLYLACTIC ACID BY PLA
DEPOLYMERASE ISOLATED FROM THERMOPHILIC
BACILLUS:
 In this study, the thermophile isolatedfrom plastic rich
environment was a source of purificationand
characterizationof PLA Depolymerase. P-nitro phenyl
acetate was used as a substrate to check the esterase
activity of PLA Depolymerase.
 The esterase activity was started from day 5, reached
maximum on day 20 and become limited at later stages
of degradation.
 PLA Depolymerase was purified with a molecularmass of
44KDa from thermophilic bacterium, Bacillus
licheniformis. The PLA Depolymerase hasoptimal activity
around 50-60Ç at pH of about 6-7.
 The activity of PLA Depolymerase at 50-60Ç shows its
thermophilicnature. Its activity was studied in the
presence of solvents at concentrationof 5% v/v.
 Ethyl acetate inhibitedits activitywhile other solvents
like ethanol, methanol,acetone, DMSO, hexane,
isopropanoland acetonitrileshows a minorinhibitionin
its activity.
 Therefore it can be concludedthat enzyme derived from
B.licheniformis was an esterase but it is different from
other esterases depending upon theirtemperature, pH
and molecular weight.

24. ISOLATION OF THERMOPHILES DEGRADING POLY
LACTIC ACID:
 A thermophile, which degrades Polylactic acid at 60Ç was
isolated and characterized from 153 soil samples with an
enrichment culture technique.
 At this temperature the strain grew on PLA and shows a
change in the total organic carbon concentration in the
medium. As for residual PLA the viscosity number
decreased rapidlybut weight decreased slowly.
 The strain 41 was identifiedas a neighboring species to
Geobacillus thermocatinulataus, which had an optimum
growth temperature of about 60Ç. It would be the first
thermophiledegrading PLA from the genus Geobacillus.

25.  In the absence of strain decrease in weight of PLA was
hardly observed.
THERMOPHILIC ANAEROBIC BIODEGRADATION
OF PLA:
 The anaerobicbiodegradation ofPLA was observed at
55Ç using the evaluationsystem reported previously
(modified ISO 13975 method). The biodegradabilityof
PLA was 75 % in 75 days. Some bacteria that participated
in anaerobicdegradationwere of unique character.
 Anaerobic degradationof PLA was done by an organism
with 100% identicalto Ureibacillus sp. It was also done by
other three organisms with poorsimilarity to that specie.
Bacillus infernus, Propionibacterium sp., and two
unidentifiedspecies were used in the anaerobic
biodegradation ofbiodegradableplastics.
 Anaerobic degradationalso produces CH4 as an energy
resource. Thermophilicanaerobicfermentation plants
that operate at 55Ç are currently working in Japan. By
applyingthis method, poly lactic acid (PLA) powder was
biodegradedby approximately60% in 30days, 80% in
40days, and 90% in 60 daysat a total solid concentration
of approximately 2% in the sludge.
 In this method, RT-PCRDGGE techniques are appliedto
sludge containingseparately introducedcommercial

26. bioplastics.This is done to identify eubacteria
participatingin the anaerobic biodegradation.The
biodegradability% was calculated by using following
equations:
CRITICAL ANALYSIS:
 The degradationof PLA is enhanced by the increase in
temperature and relative humidity.
 Its high molecularweight. Poly lactic acid lose its tensile
strength when its molecularweight is in the range of
50,000-75,000 g/mol.
 Its mechanicalproperties also lost at high temperature
which is achievableat thermophilic conditions.

27.  Therefore PLA degraded more efficiently at thermophilic
conditions(i.e.55Ç) than at mesophilic conditions.
DISCUSSION AND CONCLUSION:
 Different methods and approaches are used to degrade
Polylactic acid by using thermophilicconditions.
 Thermophilicbiodegradation ofPolylactic acid is done
using different methods like MODA-B, Thermogravimetric
analysis, PLA Depolymerase activity and others.
 All these approaches suggest that PLA is degraded at high
temperatures. Low temperature affects its degradability.

28. REFERENCES: