**6. Biochip analysis of environmental samples for the presence of PHA-producing bacteria**

A low-density biochip as a collection of DNA probes arranged on a solid matrix was the first time applied for rapid screening of PHA-producing organisms in the environment. This technique performs detection using a single undivided environmental sample and allows the identification of different genes in one reaction. The development of a biochip includes the following steps: design of the appropriate probes; equalization of the hybridization capacity of the probes for their inclusion in the biochip prototype; environmental DNA preparation for hybridization; and the fine-tuning of a biochip for the optimization conditions of bacteria detection.

Eleven oligonucleotide probes for the *phaC* genes responsible for the metabolic pathways involved in PHA production were designed (**Table 4**). The phaC gene codes


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


#### **Table 4.**

*Probes used in the biochip.*

a subunit common to the four known PHA synthase classes (class I, class II, class III, and class IV) [35], which are found in different species of bacteria; the PHA C polypeptide subunit varies in molecular weights. The BLAST search in the NCBI database (http://www.ncbi.nlm.nih.gov) was used to derive the sequences used for the probe design. The complete nucleotide sequence for the genomic DNA of PHA-producing microorganisms was downloaded and saved in FASTA format. The sequence was loaded into the SeaView software, which is a sequence alignment editor allowing manual or automatic alignment through an interface with the CLUSTALW program. Region variations in the sequences were identified from multiple alignments generated by the Clustalo option in SeaView [36] or creating multiple alignments of protein sequences by Muscle [37]. Based on final alignments, conserved parts of the sequences of each gene were chosen and used for probe designing using the OLIGO 7 program [38]. The phylogenetic tree generated after a distance-based analysis using the Kimura 2 model and neighbor-joining algorithm showed a clear distinction between the classes of *phaC* genes. After the phylogenetic analyses, the probes were designed to cover different classes of PHA synthase.

Probes (4–10) target PHA synthase (*phaC*) gene, probes (2,3) target poly-betahydroxybutyrate polymerase gene, and probes 1 and 11 target both genes.

The 3D dendrimeric matrixes for biochip preparation were manufactured at Tbilisi State University, Andronikashvili Institute of Physics, Georgia. All procedures concerning dendrimeric matrix activation for probes immobilization, and dendrimeric matrix deactivation for environmental DNA hybridization are presented in our previous publication [39].

In order to check and evaluate that the chosen probes have similar hybridization capacities, the cassette approach developed and published in Ref. [40] has been used. If the probes exhibit the same hybridization characteristics, they should give equal

fluorescent signals in hybridization reactions. The result of the estimation of the hybridization capacity of the proposed probes showed that the probes exhibited comparable hybridization signals and were characterized by the mean signal-to-noise ratio S/N = 12 (data not shown). These probes were tested against DNA obtained from environmental samples (sludge and compost) samples. Total DNA was purified from the environmental samples (sludge or compost) using MO BIO PowerSoil® DNA Isolation Kit (MO BIO technologist, Inc.). The procedures for DNA preparation for hybridization that include DNA amplification, fragmentation, and fluorescent labeling are described in Ref. [40]. Two sludge and three compost environmental samples were analyzed for the bacterial assemblage, revealing PHAs producing capacity using a biochip method. **Figure 7** represents the proportion/interrelation of the issued by the probes´ bacterial species in the functional assemblage of sludge T2, as the equalized biochip is the basis for the estimation of the microbial ratio. The hybridization signal S/N above 1.5 was counted as a reliable result.

According to **Figure 7** and **Table 4**,


#### **Figure 7.**

*The results of DNA from T2 sludge sample hybridization with the biochip. Panels A and B represent the arrangement of the probes on the biochips. The red circles and PCM are the position control markers. The white circles mark the position of the reliably visible signals detected after hybridization with the probes for phaC gene. Panel C shows the hybridization signal, presenting a signal-to-noise ratio, for the probes on the biochip. The data presented are mean values SD.*

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

#### **Figure 8.**

*The results of DNA from T5 compost sample hybridization with the biochip. Panels A and B represent the arrangement of the probes on the biochips. The red circles and PCM are the position control markers. The white circles mark the position of the reliably visible signals detected after hybridization with the probes for phaC gene. Panel C shows the hybridization signal, presenting as a signal-to-noise ratio, for the probes on the biochip. The data presented are mean values SD*.

Therefore, the bacteria species present in sample T2 are *Synechococcus*; *Pseudomonas*; *Cupravidus,* and *Aquabacterium*. The first three species are very well-known PHA producers.

**Figure 8** represents the proportion of the bacterial species in the functional assemblage of compost T5.

According to **Figure 8** and **Table 4**,


Therefore, all studied types of bacteria are present in sample T5. Dominant bacteria species in sample T5 are *Synechococcus*, *Pseudomonas*, and *Cupravidus*, well-known PHA producers.

**Table 5** summarizes the result obtained with biochips for the studied environmental samples.

The compost samples T5 and T6 contain all studied bacteria species with *phaC* gene. The bacteria spp. *Synechococcus* and *Cupravidus* (probes number 5 and 10) were detected in all environmental samples (sludge and composts). The absence of hybridization signals on the biochip for some bacteria might be explained by the absence of these bacteria in the samples.

*Advances and Challenges in Microplastics*


**Table 5.**

*Summary of the phaC gene detected in the biochip from environmental samples.*
