**3. PCR amplification of PHAs genes**

The genomic DNA from the 48 strains that tested positive in the SBB staining test was analyzed for the presence of the PHA synthase gene (phaC) using the PCR amplification method. Genomic DNA was recovered using the DNeasy Blood and Tissue kit (Qiagen®) according to the manufacturer's instructions. The phaC gene was amplified using F I-179 L forward primer and R I-179R reverse primer (**Table 1**) [24]. Final reactions contained: 2x GoTaq® Green Master Mix and 10 μM of each oligonucleotide primer. PCR was carried out as indicated: initially denatured at 94°C for 10 min; followed by 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min followed by the final extension step at 72°C for 10 min.

The phaC gene was also amplified using the PhaCF1BO forward primer and PhaCR2BO reverse primer (**Table 1**) [25]. Final reactions contained: 2x GoTaq® Green Master Mix and 10 μM of each oligonucleotide primer. PCR was carried out as indicated: initially denatured at 95°C for 10 min; followed by 30 cycles of 95°C for 1 min, 57°C for 45 sec, and 72°C for 2 min followed by the final extension step at 72°C for 5 min.

**Primer Sequences of primers 5**<sup>0</sup> ! **3**<sup>0</sup> **Gene target Size of amplicon, bp** 8F AGAGTTTGATCCTGGCTCAG *16 S rRNA* 1500 1492R GGTTACCTTGTTACGACTT F I-179L ACAGATCAACAAGTTCTACATCTTCGAC *phaC & phbC* 540 R I-179R GGTGTTGTCGTTGTTCCAGTAGAGGATGTC PhaCF1BO TCMYCTSKACTGCSCTGGYG *phaC* 247 PhaCR2BO YWGCTRGACYAGACCTGGAT

The selective results of the amplification using the pair of primers F I-179 L/R I-179R are presented in **Figure 4**. The isolates tested positive for the presence of the

#### **Table 1.**

*Oligonucleotide primers are used in PCR.*

#### **Figure 4.**

*The DNA amplicons of phaC gene (540 bp) with primers: F I179L/ R I179R from the bacterial isolates. Panel A: Lane 1: 1 kb DNA Ladder; Lane 2: 3/1; Lane 3: 10/1; Lane 4: 12/1; Lane 5: 17/1; Lane 6: 19/1; Lane 7: 20/1; Lane 8: 22/1; Lane 9: 25/1; Lane 10: 100 bp DNA Ladder; Lane 11: 9/1; Lane 12: 6/2; Lane 13: 8/2; Lane 14:10/2; Panel B: Lane 1: 1 kb DNA Ladder. The isolates presented in the Lanes 2 to 13 are as follows: Lane 2: 13/ 2; Lane 3: 16/2; Lane 4: 17/2; Lane 5: 18/2; Lane 6 to 9 negative results for isolates 2/1, 4/1, 3/2, 14/2; Lane 10: 9/2; Lane 11: 19/2; Lane 12: 24-1/2; Lane 13: 24/2.*

#### **Figure 5.**

*The DNA amplicons of phaC gene (247 bp) with primers: PhaCF1BO/PhaCR2BO from the bacterial isolates. Panel A: Lane 1: 1 kb DNA Ladder; Lane 2: 3/1; Lane 3: 10/1; Lane 4: 12/1; Lane 5: 17/1; Lane 6: 19/1; Lane 7: 20/1; Lane 8: 22/1; Lane 9: 25/1. Panel B: Lane 1: 1 kb DNA Ladder. The isolates presented in the Lanes 2 to 12 are as follows: Lane 2: 9/1; Lane 3: 6/2; Lane 4: 8/2; Lane 5: 10/2; Lane 6: 3/1; Lane 7: 10/1; Lane 8: 12/1; Lane 9: 17/1; Lane 10: 19/1; Lane 11: 20/1; Lane 12: 22/1.*

*phaC* gene in case in their DNA correct amplicon size (540 bp) was revealed when run on the 1.2% agarose gel (**Figure 4A** and **B**).

The selective results of the amplification using the pair of primers PhaCF1BO/ PhaCR2BO are presented in **Figure 5**. According to **Figure 5**, some of the isolates tested positive for the presence of the *phaC* gene in their DNA as shown in lanes (3, 4, 6, 7, and 9) (**Figure 5A**) and lanes (2, 7, 9, 11, and 12) (**Figure 5B**). These isolates produce the correct amplicon size (247 bp) when run on the 1.2% agarose gel.

*Pseudomonas oleovorans* NCTC 10692 (Bacteria Collection from Public Health England, ATCC 8062), which is the producer of PHA [26, 27] was used as a positivecontrol strain. *Escherichia coli* XL-1 Blue (Agilent Technologies), which is not able to produce PHA, was used as a negative control strain.

Out of the tested 48 strains, 18 strains tested positive for the presence of the *phaC* genes confirmed by one or both types of PCR and by the presence of PHA granules confirmed by the Nile Blue A and Nile Red staining methods. These 18 strains, in addition to two strains (9/1 and 9/2) that tested negative with PCR and positive with Sudan Black B were sent for sequencing after cloning their 16S rRNA PCR products into competent *E. coli* cells (**Table 2**). The 16S rRNA gene was amplified using the primer pair 8F and 1492R (**Table 1**) [28], the conditions were pointed in Ref. [29].


*Bioplastics against Microplastics: Screening of Environmental Bacteria for Bioplastics… DOI: http://dx.doi.org/10.5772/intechopen.109756*


#### **Table 2.**

*The characteristics and sequence identification of the selected bacterial isolates from the environmental samples.*

PCR products were ligated into a vector plasmid DNA and transformed into *E. coli* competent cells using the TOPO® TA Cloning® Kit (Invitrogen by Life Technologies, UK) following the manufacturer's protocol. Sequencing was performed by GATC Biotech Ltd., London Bioscience Innovation Center, the identification was done through the BLAST search. The strains, which were isolated from sludge and compost samples, belonged to multiple bacterial families (**Table 2**), including the *Enterobacteriaceae* (7 strains, 35%), *Micrococcaceae* (2 strains, 10%), *Alcaligenaceae* (4 strains, 20%), *Moraxellaceae* (1 strain, 5%), *Aeromonadaceae* (1 strain, 5%), *Bacillaceae* (3 strains, 15%), and *Pseudomonadaceae* (2 strains, 10%). Most of the isolates from the environmental samples belong to *Enterobacteriaceae* (*Citrobacter* sp., *Raoultella*, *Klebsiella*, *Serratia,* and *Acinetobacter*), and they are commonly known as producers of polyhydroxyalkanoates [30]. The genomes of the identified isolates contain different classes of PHA synthase (I, II, and IV), producing different types of PHA based on the number of carbon atoms. Thus, *Pseudomonas* contains either class I or II of PHA synthase [12] and can produce medium-chain length PHA, but some strains are able to synthesize both SCL- and MCL-PHAs; *Alcaligenes* can produce short-chain length PHA.

### **4. Confirmation of the presence of a PHA pathway**

The isolates were screened for the presence of plasmids, as they often carry the PHA biosynthetic pathway. Plasmids were detected in all 20 selected strains for PHA production (**Table 2**). To show that the plasmid and PHA production were co-inherited, a non-PHA-producing strain of *E. coli* XL-1 Blue (Agilent Technologies) was transformed with a plasmid. This will allow the identification of plasmids that can express the *PHA* operon in *E. coli*. As a control, a known PHA-producing plasmid from *Pseudomonas oleovorans* NCTC 10692 was transformed to show that the insertion was successful. The plasmid DNA was purified from the bacterial isolates grown in culture in the LB media using the Zyppy miniprep kit (ZYMO Research, Irvine, CA) according to the manufacturer's protocol. Purified plasmid DNA was used for the transformation of *E. coli* XL-1 Blue by electroporation (MicroPulser Electroporator, BioRad). Before performing the transformation experiments, the PHA-producing strains were characterized for their antibiotic resistance profiles. This was done to identify possible determinants that could be used as counter-selective agents, assuming that it was plasmid bourn. The 20 strains were tested for their susceptibility to 16 different antibiotics, delivered in discs to check the resistance profiles of these strains grown on solid media. Four of the PHA-producing strains (6/2, 8/2, 10/2, 13/2) were sensitive to all the tested antibiotics. As all the PHA-producing strains were resistant to ampicillin and/or nalidixic acid, but the *E. coli* XL-1 Blue host strain was not, these two antibiotics were then used as a selection for the plasmid when it was transformed into the PHA-negative strain (*E. coli* XL-1 Blue). After the electroporation, transformed cells were inoculated on LB agar containing ampicillin or nalidixic acid. After overnight incubation, the colonies obtained were picked up and grown in LB medium with the appropriate antibiotics chosen (ampicillin and nalidixic acid). These cultures were then screened for PHA-producing genes by using the staining methods, PCR amplification, and sequencing. Four strains (3/1, 10/1, 17/1, and 19/1) in addition to the positive-control strain were successfully transferred into *E. coli* XL-1 Blue, and were tested positive for PHA production and the presence of the *phaC* gene. These four strains were isolated from the sludge samples collected from the Petersfield Southern Water Treatment plant. The pointed bacterial strains provide plasmids, which have the potential to synthesize PHA for industrial/commercial purposes when co-introduced into the same genetic background.
