Isolation and characterization of polyhydroxyalkanoate (PHA) producing bacteria isolate from landfill land of Kampung Jawa Banda Aceh

. The production of biodegradable plastic from microorganisms has great potential as a substitute for conventional plastic. This study aims to isolate bacterial strains capable of polyhydroxyalkanoates (PHAs) production from the Kampung Jawa landfill land (KJLL) and characterize biopolymers. The bacterial strains were able to produce PHA using a mineral salt medium (MSM) with glucose as a carbon source. The qualitative screening of PHA-producing bacteria was conducted by Sudan Black and Nile Red. Of the 64 bacteria strains, only 41 were able to accumulate PHA in Sudan Black and Nile Red. The results showed that one bacterium the Coccobacillus strain had the highest color intensity for further characterization of PHA. The characterization of PHA by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) showed a melting temperature (Tm) of 101.54 °C. X-ray diffraction (XRD) analysis revealed a crystalline structure with an index crystallinity (ICr) value of 15.82% for PHA. The results of the analysis proved that PHA was produced by bacteria isolate. This study suggests that this is the first report of the bacteria from the Kampung Jawa landfill producing PHA with good characteristics and potential biotechnology applications.


INTRODUCTION
Petroleum-based plastics have been widely used in various products for many years.Yet, plastic waste has a seriously negative impact on the environment and has accumulated due to its non-biodegradability.One of the initiatives to deal with this problem is to create biodegradable plastic, or bioplastic [1].
Bioplastic is an environmentally friendly plastic that can be degraded by microorganisms and whose components are derived in part or entirely from renewable feedstocks.This kind of plastic is aligned with the natural carbon cycle since the waste can be degraded by an aerobic or anaerobic process.On top of that, this biodegradable plastic has no toxic emissions when it is burned [2,3].
Biodegradable plastic has been developed to replace conventional plastic.In general, bioplastic can be made from microbiota, vegetable oil, and starch, but the condition and timing of its chemical structure have changed, which affects its properties and makes it degrade.Biodegradable plastic is biologically produced from renewable sources [4].
Polyhydroxyalkanoates (PHAs) can be synthesized in cells produced by microorganisms with excess carbon source and have a structure and properties very similar to conventional plastics.Polyhydroxyalkanoates have a special ability to decompose their structure naturally with the help of microbes.Another peculiarity of PHA is that it can be produced using organic waste or renewable materials as a base material, either naturally or biologically.These bioplastics-manufactured materials utilize glucose as a substrate but is synthesized from different species [5].
The properties of PHA produced from microorganisms have similarities with petrochemical polymers such as polypropylene or polyester.PHA's properties show good moisture resistance and have a distinct barrier to gases [6].The characteristics depend on the number of carbons in the monomer unit: medium-chain-length (Mcl) is very different from short-chain-length (Scl).This property of PHA is highly dependent on the biopolymer-producing host bacteria and their fermentation conditions.Scl-PHA is highly crystalline in a stereoregular polyester; the melting point ranges from 175 to 180 o C, while the glass transition temperature is between 5 and 9 o C, and the drop properties are good [1].
Polyhydroxyalkanoates (PHAs) cannot be identified without laboratory testing to confirm the bacteria as one of the bacteria containing PHAs [7].The selection of the environment to obtain PHA-producing bacteria is critical for the presence and diversity of the PHA *Corresponding Author: nazaruddin.ma@unsyiah.ac.id produced.The type of environment that is considered to contain bacteria that can synthesize PHA is an environment with high carbon sources, which can be from either waste or industrial products [8], as in dry or swampy areas.One of the sources of bacteria is landfills, due to their large amount of organic waste that has piled up for a long time.
Kampung Jawa landfill land (KJLL) in Banda Aceh is the waste disposal center of Banda Aceh.The Kampung Jawa landfill was first built in 1994 and has an area of 12 hectares.Kampung Jawa landfill land is managed in a sanitary landfill, where the discarded waste is covered with soil and compacted with heavy equipment.Then the upper layer has garbage poured onto it and then the soil is layered and compacted again.This can lead to a large amount of organic waste.A large amount of organic waste with a high carbon source content piled up in the KJLL allows bacteria capable of producing PHA to grow.

Sampling Soil
Soil samples (ST) were collected at a single sampling point with the coordinates of latitude 5 o 34'42.57o U and longitude 95 o 18'57.12o T. The sample was placed in a bottle-shaped container and transported using a cool box.

Isolation of Bacteria
The soil sample was grown aseptically in MSM medium (composition yeast extract 1 g/L; NH4Cl0 1 g/L; MgSO4 0.2 g/L; (NH4) 2FeSO4.6H2O0.01 g/L; Na2HPO4 10.2 g/L; KH2PO4 1.5 g/L; NaCl 10 g/; 10 mL trace element solution and 1 L distilled water).The trace element was composed of CoCl2.H2O 0.2 g/L; ZnSO4.7H2O0.1 g/L; H3BO3 0.3 g/L; MnCl2.4H2O0.03 g/L; NiCl2 0.01 g/L; CuSO4.5H2O0.01 g/L and 1 L distilled water).The media was divided with the required volume in the Erlenmeyer flask (media volume 1/3 of the total volume).The Erlenmeyer flask was closed with wrapped cotton gauze and aluminum foil.Then the media was sterilized [9].The sample was then incubated at 37 C, 150 rpm for 72 hrs.The inoculant was then spread on 10 Petri dishes containing a MSM medium.The isolate was allowed to grow and then transferred to an MSM medium.The colonies were transferred in duplicates of solid MSM medium and incubated at 37 o C for 72 hrs.A single bacteria colony was screened by the dilution method.The serial dilution of isolation bacteria was prepared around a total dilution factor of 10 -5 µl/mL.The bacteria were identified with color testing Sudan Black and Nile Red.

Qualitative Screening of PHA Bacteria
The identification of bacteria isolates in producing PHA was determined by growing the isolates onto an agar MSM medium containing Sudan Black and Nile red.The grown bacteria isolates were added Sudan Black 0.02% and then washed with 70% ethanol for identification of PHA production.Black and granular pigment marks suggested that the isolates can accumulate PHA.Then Sudan Black in MSM medium was screened with Nile Red (0.25 mg Nile Red added per mL Dimethylsulfoxide (DMSO).The isolate was inoculated onto the medium for 72 hrs, then the colony was observed below Ultra Violet (UV) rays.Screening of Sudan Black and Nile red is required to identify the PHA production of bacteria.

Morphological and Gram Staining Isolate
Bacteria isolate was regenerated on an MSM medium.Morphological characterization of the bacterial isolate was determined such as size, shape, color, and texture.The bacteria staining procedure was conducted according to the previous study by Hidayat et al., 2022 [10].To determination of Gram staining, bacterial isolates were taken and smeared onto sterile preparations, which were then fixed.One drop of violet crystal was applied to the surface of the bacterial layer and left for 3 minutes.After 1 minute, the preparation was then rinsed with 96% alcohol until the dye faded, followed by a water rinse and drying with spiritus.Once dry, a drop of Lugol solution was added to the preparation and allowed to stand for 1 minute, followed by a 45-second exposure to one drop of safranin.The preparation was then viewed using a microscope at 100x magnification, and if a blue/purple color was observed, the result of Gram staining from the isolate showed that the isolate was Gram-positive bacteria.

Growth Curve PHA Study
A 250 mL Erlenmeyer flask containing 50 mL of MSM medium was inoculated with a single colony isolate and incubated at 37°C with constant stirring at 150 rpm for 24 hrs.Then the bacterial culture was inoculated in the production medium in a 500 mL Erlenmeyer Flask.The medium was then incubated at 37 o C, 150 rpm for 72 hrs to study the growth of the isolate and PHA production.The growth of the bacteria isolate was measured in 12-hrs intervals using spectrophotometry at 600 nm.Data were obtained from the absorbance measurement results plotted as OD600 versus incubation time.

Extraction and PHA Production
PHA was extracted from 20 mg of dried biomass by 1 mL chloroform (99% v/v) and 1 mL sodium

Characterization of Extracted PHA
The isolated PHA was characterized with a Fourier Transform Infra-Red (FTIR, PerkinElmer, USA), Scanning Electron Microscope (SEM, FEI NOVA Nano SEM 230), X-ray Diffractometry (XRD, PANalytical Xpert PRO), Differential Scanning Calorimetry (DSC,) and Thermogravimetric Analysis (TGA) from Mettler Toledo's.FTIR analysis was used to detect functional groups of PHAs.The spectrum was analyzed by FTIR at wave numbers from 500-4000 cm - 1 .SEM analysis observed the morphological surface of the with 20 kV and 8 mA at 200x magnification.XRD analysis was performed to determine the crystal phase and size of the PHA produced.Samples were scanned from 2θ = 0° to 65°.The data was processed with the use of Origin software (Origin 8.5, USA).To determine the melting point (Tm) of PHA, DSC analysis was conducted.Meanwhile, TGA analysis was carried out to evaluate the material weight changes and their rate over time or temperature under an inert atmosphere.

Bacterial Isolation
The sampling soil sample of the Kampung Jawa landfill had a pH of 7.2 where it was known that the pH of the sample could affect bacterial growth [11].Bacteria was grown in a liquid media, which is distinguished by the change in color of MSM media from clear yellow to turbid yellow.This research successfully obtained as many as 210 colonies of bacterial isolates.These results indicated that bacteria sampled from Kampung Jawa landfill were grown on solid and liquid MSM media.

Identification of PHA-Producing Bacteria
The Sudan Black and Nile Red Tests were used to identify bacteria that produce PHA.The test was a qualitative screening to determine whether the obtained bacterial isolates produced PHA.The results of the Sudan Black and Nile Red tests with high intensity were a group of bacteria that had a good ability to produce PHA.The appearance of black on the isolate indicated the Sudan Black method test (Figure 1a).The Sudan Black test obtained 64 isolates that were able to accumulate PHA from 210 bacterial isolates.This result showed that the bacteria from landfill land of Kampung Jawa had a score of 30.48% for PHA production.Mohammed et al., (2019) [12] reported that the staining of bacterial isolates obtained with Sudan Black identified as many as 20 bacterial isolates from samples taken from plastic waste fields.The isolates obtained were from the groups Bacillus sp.BPPI-14 and Bacillus sp.BPPI-19, which when dyed using Sudan Black produce black grains.All isolating stains showed positive results on PHA screening.Another study conducted by Evangeline & Sridharan., (2019) [13], obtained about 30% of the bacteria from soil isolated from different types of regions using the Sudan Black method.A total of 30 isolates had positive Sudan Black dye results, indicating the presence of PHA isolates, which were indicated by the presence of PHApositive bluish-black grains and isolates that did not indicate the presence of negative bluish-black grains.The study conducted by El-malek et al., (2020) [14] obtained as many as 122 bacterial isolates tested with Sudanese Black staining obtained black grains which indicated PHA positive in as many as 12 isolates.However, the Sudan Black test was less selective to   [15] reported that they successfully isolated 9 bacteria from the soil of the marine environment.Maheshwari et al., (2018) [16] also got one strain that is brighter in color or more prominent than others.A similar study was also conducted by Ng & Sudesh., (2016) [17] where one strain was obtained that had a lighter color than the other strains.Another study reported by Cai et al., (2021) [18] which produces PHA using silkworm waste as a carbon source obtained 4 endogenous microorganisms that appeared when observed with Nile Red staining.This occurs because of the accumulation of PHA in strains of bacteria tested using the Nile Red method, which shows better fluorescence compared to other strains.Based on the results, one isolate that was both positive in the Sudan Black test and had a high intensity on the Nile Red test was selected for morphological assay, Gram staining, production, and characterization of PHA.

Gram Morphology and Staining Test
The isolates of PHA-producing bacteria obtained in this study (Figure 2a).Based on morphology and Gram staining isolate, the isolate showed as a round shape, overall edges, convex elevation, white in color, and Gram-positive.Bacterial isolates when staining Gram with violet crystals was known to retain the purple color (Figure 2b).These results suggest that the bacteria was Gram-positive with single strain [19][20].
The results of the morphology study analysis suggested that the isolate containing Coccobacillus cells.Coccobacillus is a bacterium with a cellular morphology that is intermediate between Cocus and  Bacillus.The bacterium in this group is known to be one of the bacteria that produces PHA [21].

Growth Curve of PHA-Producing Bacteria
The bacterial growth curve in the PHA production was obtained at 37 o C, 150 rpm for 72 hrs incubation time.The growth curve with the number of bacterial cells was calculated for 72 hrs at an interval of 12 hrs with a UV-Vis spectrophotometer at λ 600 nm expressed as the optical density value (OD600) (Figure 3).
There were 2 phases of the bacterial growth curve in PHA production onto medium, namely the log phase and the stationary phase and there was no lag phase.
The bacteria have been grown on the production medium for 24 hrs so that they became adapted.The logarithmic phase of the bacterium at intervals of 0 to 36 hrs was indicated by the increased growth of bacterial cells.At 36 to 72 hrs, the bacteria entered in to the stationary phase.It was shown that there was a slight increase in the growth of the bacteria.Bacteria was known to be able to produce PHA as a secondary metabolite optimally in the stationary phase [22].The stationary phase of growth and production of PHA by Bacillus sp at 45 to 72 hrs time.Based on the growth curve, this study proved that PHA was produced at an incubation time of 36 hrs with glucose-containing MSM medium of the bacteria from landfill land of Kampung Jawa.

PHA Production and Extraction
PHA production using glucose as a carbon source was obtained as much as 0.165 g of PHA solids.The yield from the production medium was 4.9% (0.034 g/L).The yield of this study was smaller than the yield of PHA production from Bacillus subtilis using cooking oil waste as a carbon source 38.4% (12.3 g/L) [23].This was thought to be due to its carbon source composition, suboptimal growth, purification process, and PHA production [24][25][26][27][28].

PHA Characterization
The PHA of isolation results in this study were further characterized by Fourier Transform Infra-Red, X-ray

Determination of Functional Groups of PHA by Fourier Transform Infrared (FTIR) Analysis
Polyhydroxyalkanoates was characterized using FTIR to determine the functional groups in PHA from isolates [29].PHA is a polymer that has functional groups of consisting of carbonyl compounds, esters, and aliphatic carbon chains.Each of the functional groups can be given a distinctive spectrum to a specific wave number in the FTIR spectrum.The results of the FTIR analysis showed that the PHA from KJLL isolate were of an O-H stretching vibration of the hydroxy group at wave number 3416 cm -1 , and the vibration of the C-H group of alkanes at wave number 2932 cm -1 .Furthermore, the C=O stretch vibration of the carbonyl ester was identified at wave number 1746 cm -1 and wave number 1638 cm -1 .In the isolates, the vibration of the C-H group of the methyl functional group was found at wave numbers 1440 cm -1 , and C-O-C cm -1 at wave numbers 1248 cm -1 and 1083 cm -1 , the four main compound groups showed distinctive characteristics of PHA [30,31,23].
The characterization results in this study were close to the range of wave numbers from PHA that has been reported by several previous studies which can be seen in Table 1.

Determination of PHA Structure by X-ray Diffraction (XRD) and Scanning Electron Microscope (SEM) Analysis
The XRD characterization result was to determine the crystalline phase of the PHA produced from bacteria isolate with glucose as a carbon source.The XRD pattern of PHA was shown in Figure 5.The diffractograms of PHA have four main peaks at 28.4 o ; 31.6º;45.3º; 56.3º.The XRD chromatogram was indicated a feature of the PHA from the KJLL isolate.The results were also supported by previous studies, where 4 similar peaks were obtained [30,34].The results of characterization with XRD showed the crystallinity index value of PHA obtained by 15.82%.Sedlacek et al., (2019) [35] reported that a microorganism's cell can store PHA in amorphous rather than crystalline forms.This condition was due to the amorphous form having a structure which was more elastic and flexible.During the production and extraction process of PHA, it was converted from an amorphous form to a crystalline form.This structure was also supported by the results of SEM characterization in Figure 6.
Based on Figure 6 shows that the PHA surface was shaped like a blob/grain.The morphological form obtained was uniform and interrelated between one particle and another.The characterization results of this study were supported by the research of [36] where PHA produced from the Mediterranean Haloferax was found to have a dense granule/lump-like structure.
Thermal Properties of PHA by Differential Scanning Calorimeter (DSC) and TGA Analysis Characterization using DSC was carried out to determine the thermal properties and resistance of a material in a certain temperature range.Based on Figure 7, it can be seen that the emergence of an exotherm peak at a temperature of 78.28 o C which indicates the glass transition point of the PHA, to 84.63 o C which also indicates the occurrence of a crystallization process that releases heat (Tc).Meanwhile, the next peak that appears endothermically at a temperature of 101.54 o C was Tm which indicates the occurrence of a melting process in PHA [37].This result was also confirmed by TGA analysis (Figure 8) that the Tm value of the PHA at 101. 54 o C. Rebocho et al., (2019) [38] reported that mcl-PHA was produced by Pseudomonas citronellolis from apple pulp waste with Tm 53 o C. The Tm PHA value obtained in this study was higher than by Pseudomonas citronellolis.In addition, the thermal properties were lower than in previous studies using E. coli bacteria and molasses with Tm 129.59 o C and sucrose Tm 159 o C as carbon sources [39].It is worth to note that the difference in thermal properties may be related with the carbon sources used in the production of PHA and molecular weight (Mw) of PHA.These finding confirmed the thermal properties of PHA from of the KJLL isolate with interesting properties.

CONCLUSION
The bacteria isolat from Kampung Jawa landfill land within the genus of Cocobacillus was able to produce Polyhydroxyalkanoates (PHA) as biodegradable plastic.Polyhydroxyalkanoate had a characterization structure such as crystallinity properties, a surface shaped like a blob/grain and thermal properties of Tm 101.54 o C. In addition, molecular weight for PHA needs to be characterization further.
Copyright © 2023 Jurnal Natural p-ISSN: 1411-8513; e-ISSN: 2541-4062.Open access under CC-BY license hypochlorite (5% w/v).Afterward, the sample was shaken at 30 o C, 200 rpm for 1 hr.The residual bacteria cell was removed by centrifugation at 10.000× g, for 5 min at 37 o C. Phase organic /PHA extract was added by methanol cold (95%v/v) as much as 4x the volume of the extract.The solution was stirred to produce sediment.The sediment was stored at a temperature of 0 o C-4 o C for 24 hrs.The PHA precipitate was filtered and subsequently dried in an oven at 30 °C for 24 hrs.PHA was then weighed and stored for analysis.The % yield of the PHA generated was calculated with equation (1).%yield = !"!# × 100%..........(1) Where: m1 was PHA weight; m0 was the weight of the dried cell.

Figure 1 .
Figure 1.(a) Detection of PHA producing bacteria with Sudan Black and (b) detection of PHA producing bacteria with Nile Red.

Figure 2 .
Figure 2. Morphology characteristic of the bacteria from landfill land of Kampung Jawa (a) Morphology of bacteria, (b) Gram-Positive staining bacteria on microscopes.

Figure 3 .
Figure 3. Growth curve for PHA-producing of the bacteria from landfill land of Kampung Jawa.

Figure 4 :
Figure 4: PHA isolated FTIR spectrum from soil sample bacteria in kampung Jawa landfill.

Table 1 .
Comparison of functional groups characterization using FTIR