Biodegradability of polyethylene by bacteria and fungi from Dandora dumpsite Nairobi-Kenya
Biodegradability of polyethylene by bacteria and fungi from Dandora dumpsite Nairobi- Kenya
Christabel Ndahebwa Muhonja 0 1
Huxley Makonde 1
Gabriel Magoma 0 1
Mabel Imbuga 1
0 Pan African University Institute of Science, Technology and Innovation, Nairobi, Kenya, 2 Department of Pure & Applied Sciences, Technical University of Mombasa, Mombasa, Kenya, 3 Jomo Kenyatta University of Agriculture and Technology , Nairobi , Kenya
1 Editor: Pankaj Kumar Arora, Babasaheb Bhimrao Ambedkar University , INDIA
This study aimed at isolating and identifying bacteria and fungi with the capacity to degrade low density polyethylene (LDPE). The level of biodegradation of LDPE sheets with bacterial and fungal inoculums from different sampling points of Dandora dumpsite was evaluated under laboratory conditions. Incubation of the LDPE sheets was done for sixteen weeks at 37ÊC and 28ÊC for bacteria and fungi respectively in a shaker incubator. Isolation of effective candidates for biodegradation was done based on the recorded biodegradation outcomes. The extent of biodegradation on the polyethylene sheets was assessed by various techniques including weight loss analysis, Fourier Transform Infrared Spectroscopy (FTIR) and GC-MS. Fourier Transform Infra-Red spectroscopy (FTIR) analysis revealed the appearance of new functional groups attributed to hydrocarbon degradation after incubation with the bacteria and fungi. Analysis of the 16S rDNA and 18S rDNA sequences for bacteria and fungi respectively showed that bacteria belonging to genera Pseudomonas, Bacillus, Brevibacillus, Cellulosimicrobium, Lysinibacillus and fungi of genus Aspergillus were implicated as polyethylene degraders. An overall analysis confirmed that fungi are generally better degraders of polyethylene than bacteria. The highest fungal degradation activity was a mean weight reduction of 36.4±5.53% attributed to Aspergillus oryzae strain A5, 1 (MG779508). The highest degradation activity for bacteria was a mean of 35.72± 4.01% and 20.28± 2.30% attributed to Bacillus cereus strain A5,a (MG645264) and Brevibacillus borstelensis strain B2,2 (MG645267) respectively. Genus Aspergillus, Bacillus and Brevibacillus were confirmed to be good candidates for Low Density Poly Ethene bio-degradation. This was further confirmed by the appearance of the aldehyde, ether and carboxyl functional groups after FTIR analysis of the polythene sheets and the appearance of a ketone which is also an intermediary product in the culture media. To improve this degrading capacity through assessment of optimum conditions for microbial activity and enzyme production will enable these findings to be applied commercially and on a larger scale.
Data Availability Statement: All relevant data are
within the paper.
Funding: This research is supported by the African
Union Commission (ADF/BD/WP/2013/68 to CNM)
and the Japanese International Co-operation
Agency (JICA) (00025 to CNM). The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
Approximately 140 million tons of plastics are produced every year and high amounts find
themselves in the ecosystem as industrial waste products [
]. About 30% of the plastics are
used worldwide for packaging of foods, pharmaceuticals, cosmetics, detergents and chemicals
and this is still expanding at a high rate of 12% p.a [
]. Plastics have replaced paper and other
cellulose-based products for packaging because they have better tensile strength, lightness,
resistance to water and microbial attack. Commonly used plastics have been categorized as
polyethylene (LDPE, MDPE, HDPE and LLDPE), polypropylene (PP), polystyrene (PS) and
polyvinyl chloride (PVC) [
]. Low Density Polyethylene belongs to thermoplastics class [
and is believed to have non-degradable nature due to its hydrophobic backbone [
]. This has
forced many governments to come up with measures to curb this menace. Bangladesh, for
instance, imposed a ban on plastic bags in March 2002 following flooding caused by blockage
of drains. The management of solid waste in Kenya has been poor and even worst in major
cities such as Nairobi [
]. The consumption of plastics in the country has increased to 4,000 tons
per annum of polyethylene bags which together with hard plastics end up scattered in the
environment creating ªthe plastics menaceº. Kenya through the National Environmental
Management Authority (NEMA) has embraced the 3Rs: Reduce, Re-use and Recycle concept of solid
waste management but this has not addressed the problem of polyethylenes which remain
scattered in the environment as recorded by [
]. Most recently, the Kenyan ministry of Natural
Resources, through the NEMA imposed a ban on the use of polyethylene carrier bags from
28th August 2017 in an attempt to reduce the amount of polyethylene being released into the
]. Despite this move, polyethylene continues to be used especially in packaging
Biodegradation can be defined as the decomposition of substances through microbial
activity. This is a complex process which involves several steps [
]: bio-deterioration (the combined
action of microbial communities and abiotic factors to fragment the materials into tiny
fractions), depolymerization (Microorganisms secrete enzymes and free radicals able to cleave
polymer into oligomers, dimers and monomers, assimilation (some molecules are recognized
by receptors of microbial cells and can go across the plasma membrane) and mineralization
(simple molecules as CO2, N2, CH4, H2O and different salts from intracellular metabolites that
are completely oxidized are released) [
]. Bacteria and fungi have been implicated in this
process albeit slow rates. According to [
], Pseudomonas species are most highly implicated in the
biodegradation of LDPEs. They isolated Pseudomonas citronellolis EMBS027 which had 17.8%
weight reduction on polyethylene sheets. [
] isolated Brevibaccillus borstelensis strain 707
which upon 30 days incubation at 50ÊC reduced the gravimetric and molecular weights of
polyethylene sheets by 11 and 30% respectively. Fungal isolates: Fusarium sp. AF4, Aspergillus
terreus AF5 and Penicillum sp. AF6 were found attached to Polyethylene sheets mixed with
sewage sludge for ten months [
]. Ability of Bacillus subtilis to degrade polyethylene was also
demonstrated by [
] in the presence and absence of bio-surfactants. [
] isolated several
fungal genera that were able to degrade polyethylene sheets with Aspergillus niger showing the
highest weight reduction of 4.32%.
Fourier Transform Infrared spectroscopy (FT-IR) is used to indicate the map of the
identified compounds on the surface of the sample and document via collection of spectra [
Spectra of sheets obtained from four different LDPE samples by [
] showed introduction of some
new peaks after the period of biodegradation with peaks of carbonyl groups (1720 cm-1), CH3
deformation (1463 cm-1) and C = C conjugation band (862 cm-1). The weight loss, percentage
of elongation and change in tensile strength can be applied to measure the physical changes of
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the polyethylene. The products from polyethylene degradation are also characterized using
techniques such as Gas Chromatography-Mass Spectrometry (GC-MS) [
This study aimed at isolating and identifying bacteria and fungi that have the capacity to
degrade low-density polyethylene. Evidence of biodegradation was based on weight loss,
FT-IR and GC-MS outcomes which confirmed that microbes are capable of degrading LDPE.
Materials and methods
Dandora dumpsite is about 8 km away from Nairobi city and is adjacent to the heavily
populated low income estates of Dandora, Korogocho, Baba Dogo and Huruma. It is home to a 1\4
a million people and it sits on over 30 solid acres. It contains mixed wastes that include
domestic wastes from homes, expired goods, agricultural wastes, and hospital wastes most of which
are or come in polyethylene carriers. Polythene bags made up 225 tons of the 2000 tons of
waste in Nairobi in a single day by the year 2006. The sampling points were as follows: A:
S01Ê14.633 E036 Ê54.063 Elevation 1578m, 21.48Km SW, B: S01Ê14.652 E036 Ê54.031
Elevation 1589m, 21.55Km SW, C: S01Ê14.661 E036 Ê53.977 Elevation 1590m 21.63km SW D:
S01Ê14.688 E036 Ê53.986 Elevation 1594m 21.65km SW and E: S01Ê14.710 E036 Ê53.977
Elevation 1596m 21.69km SW
Sample collection and preparation of medium for LDPE degrading bacteria
A randomized block design was used to identify points for sample collection. Soil samples
were collected randomly from the five selected sampling blocks of the dumpsite. The samples
from each sampling block were collected at 5 different points of 1m diameter. This resulted in
25 soil samples collected. Soil was aseptically scooped from and adjacent to buried
polyethylene materials at 5 cm depth below the litter layer. On-the-site temperature was recorded in
order to ascertain the in-situ biodegradation conditions. Samples were kept in Ziploc bags and
transported to the Institute of Biotechnology Research (IBR) lab at JKUAT in a cool box. Once
in the lab, the pH of the samples was also measured and recorded.
Preparation of artificial media and incubation
1 g of soil sample was added to 50ml of 0.85% autoclaved normal saline solution to prepare the
inoculums. The inoculum was kept at 37ÊC for 2±3 hr in a shaker incubator before inoculation.
Artificial media was prepared i.e. 0.1% (NH4)2 SO4, 0.1% NaNO3, 0.1% K2HPO4, 0.1% KCl,
0.02% MgSO4 and 0.001% yeast extract in 1000ml distilled water [
]. LDPE powder weighing
2g was added as the carbon source to each 100 ml of growth medium. 1% of the prepared
inoculums was transferred to 200 ml of synthetic medium to prepare growth culture for the
LDPE degrading microorganisms. Sheets of Polyethylene (app 3 cm x 3 cm each), weighed,
disinfected in 70% ethanol and air-dried for 15 min in an oven were introduced into the
synthetic media. Culture flasks for fungal incubation were augmented with 250mg/ml ampicillin
to inhibit bacterial growth. All treatments were done in triplicates. Synthetic media with LDPE
without inoculum was used as the negative control. All the treatments were incubated in an
incubator shaker at 150 rpm for up to 16 weeks.
Determination of LDPE degrading potential of the bacterial isolates
The LDPE sheets were recovered after different incubation intervals (8 weeks, 12weeks and 16
weeks). Washing of the sheets was done using 2% SDS to remove the bacterial biomass then
dried overnight before being weighed. At the end of the incubation period, the structural
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changes in the LDPE surface was investigated using the EQUINOX 55 FT-IR spectrometer [
For each LDPE sheet, a spectrum was taken from 400 to 4000 wave numbers at a resolution
of 2 cm-1 and over 32 scans. The control was not subjected to any incubation process. The
incubation media was subjected to GC-MS analysis. The results were recorded and analyzed.
Isolation of LDPE degrading microorganisms
Based on the results above, bacterial isolation was done from the culture flasks at the end of
the incubation period. Isolation was only carried out from those incubation flasks that had
shown indications of biodegradation based on weight changes, FTIR and GC-MS outcomes.
For bacterial isolation, a loopful of culture from the synthetic media was put on a nutrient
agar plate, a spreader was used to spread it till dry. Incubation was done overnight at 37ÊC or
up to three days for slow growing bacteria. The mixed cultures of bacteria were continually
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sub-cultured to obtain pure bacterial cultures. They were stored at± 20ÊC and at -80ÊC in 15%
For fungal isolation, a drop of the inoculum was put and spread till dry on Potato Dextrose
Agar (PDA) plates and incubated for five days. The various fungi were isolated based on
morphology and sub-cultured continually to obtain pure fungal cultures.
Identification of isolates
Total genomic DNA for 16S rDNA amplification was isolated from bacterial cells grown
overnight by means of a standard protocol [
]. Amplification of the 5' end of the 16S rDNA gene
was performed with universal primers forward primer (8-F) 5'-AGAGTTTGATYMTGGCTCA
G- 3' and reverse primer (1942R) 5'- GGTTACCTTGTTACGACTT-3' [
]. For fungal
isolates, CTAB method of genomic DNA extraction was used [
]. Primer pair 566-F:5'
CAGCAGCCGCGGTAATTCC - 3' and for 1200-R:5'- CCCGTGTTG
AGTCAAATTAAGC3' which amplify on average a 650 bp long fragment from the V4 and V5 regions [
used. The similarity of the sequences obtained against known deposited 16S rDNA and 18S
Means with same superscript letters in the same column are not significantly different while means with different superscript letters are significantly different using
Fisher's Protected Least Significant test at (P<0.05). The superscript letters are arranged in ascending order with `a' indicating the least significant mean change in
weight while `h' indicates the most significant mean change in weight. Means that have a shared range have a shared superscript letter.
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Fig 1. % mean weight reduction of the 30 micron polyethylene sheet incubated with 25 bacterial sub-samples
which were found to contain specific bacterial isolates after incubation at 37 ÊC for sixteen weeks. Each mean
weight represents the average of three replicates ± SE.
Fig 2. % mean weight reduction of the 30 micron polyethylene sheets incubated with 25 fungal sub-samples which
were found to contain specific isolates after incubation at 28 ÊC for sixteen weeks. Each mean weight represents the
average of three replicates ± SE.
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Fig 3. % reduction in mean weight of the 30 micron polyethylene sheets after sixteen weeks incubation with fungal
and bacterial inoculums at 28 ÊC and 37ÊC respectively. Each mean weight represents the average of five sample
means± SE. Means with same letters are not significantly different using Fisher's Protected Least Significant test at
Fig 4. % reduction in mean weight of 40 micron polyethylene sheets after sixteen weeks incubation with fungal
and bacterial inoculums at 28 ÊC and 37ÊCrespectively. Each mean weight represents the average of five sample
means ± SE. Means with same letters are not significantly different using Fisher's Protected Least Significant test at
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Fig 5. % mean weight reduction of the 30 micron and 40 micron polyethylene sheets incubated with fungal
inoculums from sampling points A, B, C, D and E at 28 ÊC for sixteen weeks. Each mean weight represents the
average of five sample means ± SE. Means with same letters are not significantly different using Fisher's Protected
Least Significant test at (P<0.05).
Fig 6. A comparison between % mean weight reduction of the 30 micron and 40 micron polyethylene sheets
incubated with bacterial inoculums from sampling points A,B, C,D and E at 37 ÊC for sixteen weeks. Each mean
weight represents the average of five sample means ± SE. Means with same letters are not significantly different using
Fisher's Protected Least Significant test at (P<0.05).
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rRNA sequences from closely related bacteria and fungi respectively was tested with BLASTN
2.2.1 upon sequence editing with Chromas Pro version 2.6.2.
Weight loss of the polyethylene sheets
Mean weight changes were computed and used to compare the effectiveness biodegradation
between fungi and bacteria as well as between the two sets of polyethylene sheets (30 and 40
microns) (Tables 1 and 2). To get the % change in weight, the formula:
Change in weight=original weight
100% Percentage change in weight
was used. Weight loss % analysis was done and represented graphically for bacterial activity on
30 micron polythene (Fig 1), fungal activity on 30 micron polythene (Fig 2), comparative
fungal and bacterial activity on 30 micron polythene (Fig 3), comparative fungal and bacterial
activity on 40 micron polythene (Fig 4), comparative fungal activity on the 30 and 40 micron
polythene (Fig 5) and a comparative bacterial activity on the 30 and 40 micron polythene
Fig 7. FT-IR spectra of polyethylene sheet from sample B1, 1 incubated with bacterial inoculum of Pseudomonas
putida strain B1, 1a (MG645383) and the control incubated at 37 ÊC for sixteen weeks.
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FT-IR (Fourier Transform Infra-Red) outcomes
Spectral figures indicating formation of new functional groups as a result of new peaks on the
polythene sheets and the control (incubated without inoculum) sheets were superimposed as
follows (Figs 7±11).
The gas chromatogram output from the sample of Aspergillus oryzae (MG779508) indicated
the compounds and their retention times (Table 3).
Based on the above indicators of Polyethylene biodegradation as shown by the structural
changes on in FTIR spectra (Figs 7±11), the weight reduction of the LDPE sheets indicated by
the mean weight changes in graphical Figs 1±6 and the formation of intermediate degradation
products in Table 3, the respective incubation flasks containing synthetic media plus inoculum
from soil were subjected to isolation of bacteria and fungi. A total of 30 bacterial isolates were
isolated. Among this 7 were Gram negative while 23 were Gram positive. A total of 26 fungal
isolates were isolated. Among these 20 were macroscopically and microscopically profiled to
belong to the genus Aspergillus while six were profiled as belonging to the genus Penicillium.
Fig 12 shows the outcome of lactophenol blue staining of some of the fungal isolates.
Fig 8. FT-IR spectra of polyethylene sheet from sample B,4,1 with bacterial inoculum of Bacillus cereus strain A1,
a (MG645253) and the control incubated at 37 ÊC for sixteen weeks.
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Fig 9. FT-IR spectra of polyethylene sheet from sample E4, 1 with fungal inoculum of Aspergillus nidulans strain
E4,1(MG779504) and the control incubated at 28 ÊC for sixteen weeks.
Subsequent identification steps were only performed on those isolates that showed significant
Identification of bacterial and fungal isolates
The similarity of the sequences obtained against known deposited 18S rRNA and 16S rRNA
sequences from closely related fungi and bacteria respectively was tested with BLASTN 2.2.1
upon sequence editing with Chromas Pro version 2.6.2 and accession numbers obtained from
NCBI GenBank as shown (Tables 4 and 5).
The bacterial inoculum of Bacillus cereus strain A5,a (MG64264) and Brevibacillus borstelensis
strain B2,2 (MG645267) produced a mean weight loss of 35.72±4.01% and 20.28±2.30
respectively on the 30 micron polyethylene sheets (Table 1) which was significantly higher than the
other bacterial samples. This is in agreement with the results recorded by [
] in which
Brevibacillus borstelensis strain 707 after 30 days at 50ÊC reduced the gravimetric and molecular
weights of polyethylene sheets by 11 and 30% respectively. The inoculum sample of
Pseudomonas putida strain B1, 1a (MG645383) gave 2.80±0.38% mean weight loss on the 30 micron
polythene. This was however a lower rate compared to the study done by [
Pseudomonas sp was subjected to LDPE biodegradation alongside three other genera of bacteria and
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Fig 10. FT-IR spectra of polyethylene powder from sample A5, 1 with fungal inoculum of Aspergillus oryzae strain
A5,1 (MG779508) and the control incubated at 28 ÊC for sixteen weeks.
it was the most effective bio-degrader. Fungal mean weight reductions was generally higher
than bacterial with the highest mean weight reduction of 36.4±5.53%, 24±3.26% and 18±2.20%
(Table 1) attributed to isolates Aspergillus oryzae strain A5,1(MG779508), Aspergillus fumigatus
strain B2,2(MG779513) and Aspergillus nidulans E1,2 (MG779511) (Table 4) respectively.
LDPE degradation by Aspergillus and Bacillus was recorded by [
]. However the mean weight
reductions per sampling point were lower which is an indication that biodegradation of
materials varies by point location depending on the microbial composition of the particular point.
Use of weight reduction as a measure of the extent of polyethylene biodegradation has been
widely accepted and used by many authors [25±27]. These outcomes are in agreement with [
who reported the ability of microorganisms to degrade virgin polyethylene.
Analysis of the polyethylene spectral figures (Figs 7±11) indicate formation of new peaks at
the region between 1700 and 1650. Also new peaks can be seen in the region between 1000 and
1100. The new peaks at 1700±1650 are indicative of formation of aldehydes and ketones which
are intermediate products of biodegradation of polyethylene. The region of increased peak
absorbance and new peaks in the 1000±1200 cm-1 region of the FTIR spectrum correlates with
primary and secondary alcohols. The main bands of the studied LDPE sheets consist of a band
situated about 2900 cm-1 assignable to CH2 as an asymmetric stretching, a band around
1461±1466 cm-1 revealing a bending deformation, and another band at 720±724 cm-1 which
indicates a rocking deformation [
]. Intensity of the bands at 1650 cm−1 increased in the
powder samples relative to the control. These results are however in contrast to the report by
] who concluded that microorganisms can only degrade chemically or physically
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Fig 11. FT-IR spectra of polyethylene powder from sample A4, 2 with fungal inoculum of Aspergillus flavus strain
A4,2 (MG779506) and the control incubated at 28 ÊC for sixteen weeks.
pre-treated polyethylene. Presence of 4, 6-Octadiyn-3-one, 2-methyl which is a ketone in
addition to 4,4-Dimethyl-2-pentene which is an alkene in the culture supernatant through GC-MS
detection can be attributed to the process of biodegradation of the polymer where ketones are
part of the intermediary products (Table 3). This was observed in just one of the samples that
had been incubated with the consortium for degradation. This outcome is in agreement with a
previous study by [
] who reported that large number of different aldehydes, ketones and
carboxylic acids were identified in smoke generated on sheet extrusion of LDPE in an extrusion
coating process. In the present study, the degraded products were determined by GC-MS
analysis. The LDPE of 30 microns was better degraded than the 40 micron one due to its lower
molecular weight while fungal rate of biodegradation was higher than bacterial biodegradation
(Tables 1 and 2).
Among the isolates identified in this study were Pseudomonas, Bacillus, Brevibacillus,
Ochrobactrum, Lysinibacillus Cellulosimicrobium and Aspergillus (Tables 4 and 5). [
Retention time (minutes)
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Fig 12. Microscopic examination outcomes from lactophenol blue staining of Aspergillus fumigatus strain B2, 2
(MG779513) with a and b indicating erect conidia and aerial conidiophores. The conidiophores are branched with
bottle-shaped phialides at the tip of the conidiophores producing spores while d is a photo of 5-day old fungal growth.
c has a simple conidiophore terminated by flask shaped phialides where spores are produced in chains at the tip end
characteristic of genus Penicillium.
that Pseudomonas is a widely implicated bacterial genus in LDPE degradation and they also
isolated a novel strain; Pseudomonas citronellolis EMBS027 as the most effective bio-degrader.
According to [
], a thermophilic bacterium Brevibaccillus borstelensis strain 707 (isolated
from soil) utilized LDPE as the sole carbon source.
NCBI Accession number
Aspergillus nidulans strain voucher MF 109
Aspergillus insuetus strain JAU1
Aspergillus flavus strain AD-Jt-1
Aspergillus nidulans strain Ya10
Aspergillus oryzae strain RIB40
Aspergillus flavus strain Ya1
Aspergillus neoflavipes strain AJR1
Aspergillus nidulans strain FGSC A4
Aspergillus terreus strain BTK-1
Aspergillus fumigatus strain T3
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NCBI Accession number
Conclusion and recommendations
The present work indicates that naturally growing soil microbes like bacteria and fungi show
great efficacy in degrading polyethylene. Generally fungi have a higher degrading effectiveness
compared to bacteria. However both fungi and bacteria showed capacity to degrade virgin
polyethylene under laboratory conditions. Fungi of the genus Aspergillus and bacteria of the
genus Bacillus had the highest capacity of degradation compared to the other genera in this
study. Further efforts to improve this degrading capacity through assessment of optimum
conditions for microbial activity so that this concept can be applied commercially and on a larger
scale are necessary. Pre-treatment of polyethylene with substances that are environmentally
friendly could also be adopted as a means to enhance polyethylene biodegradation.
Special thanks to Pauline Akoth and Johnstone Neondo for assisting with the molecular work.
Conceptualization: Christabel Ndahebwa Muhonja, Gabriel Magoma.
Data curation: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma.
Formal analysis: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma.
Funding acquisition: Gabriel Magoma.
Investigation: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma, Mabel
Methodology: Christabel Ndahebwa Muhonja.
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