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Isolation and genomic characterization of three PHA degrading bacteria from the marine environment

Elli, Giulia LU (2019) KBTM01 20191
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
The broader goal of this project was to investigate what kind of effects the accumulation of polyhydroxyalkanoates (PHA) in the marine environment might have on the microbial world. Bacteria were isolated from biofilms fouling PHA pellets placed in the sediment-water interface; the goal was to isolate PHA degraders that were also sulphate reducers. Eleven strains were isolated and for three of them genome sequencing and genomic analysis were performed. Two of them (named SRB1LM and SRB3LM) turned out to be Bacillus strains and genes involved both in PHA degradation pathways and dissimilatory sulphate reduction pathway were detected in their genomes. These two strains also grew on agar plates where the only carbon source present was PHB... (More)
The broader goal of this project was to investigate what kind of effects the accumulation of polyhydroxyalkanoates (PHA) in the marine environment might have on the microbial world. Bacteria were isolated from biofilms fouling PHA pellets placed in the sediment-water interface; the goal was to isolate PHA degraders that were also sulphate reducers. Eleven strains were isolated and for three of them genome sequencing and genomic analysis were performed. Two of them (named SRB1LM and SRB3LM) turned out to be Bacillus strains and genes involved both in PHA degradation pathways and dissimilatory sulphate reduction pathway were detected in their genomes. These two strains also grew on agar plates where the only carbon source present was PHB (poly-(3-hydroxybutyrate)); they are facultative anaerobes and their growth at 15, 30 and 37°C was studied in laboratory conditions. In a complex medium, they both reached a higher growth rate (µmax) at 37°C; in particular, SRB1LM had the highest µ at 37°C in anaerobic conditions (0.407 h-1), while SRB3LM had the highest µ at 37°C in aerobic conditions (0.849 h-1). The third bacterial genome sequenced belonged to the Exiguobacterium genus, whose known species and strains have been isolated from a variety of environments. This isolate (named SRB7LM) can also grow on PHB agar plates, it is a facultative anaerobe but does not have genes of the dissimilatory pathway of sulphate reduction. SRB7LM had a similar µ at the three tested temperatures in anaerobic conditions (0.2 h-1), which were higher compared to the ones in aerobic conditions.
The two Bacillus strains could be interesting for the future since the increase in PHA production will cause a higher accumulation in the marine environment; this, coupled with the already high concentration of sulphate, could arise favoring conditions for their growth. (Less)
Popular Abstract
Isolation and genomic characterization of three PHA degrading bacteria from the marine environment.
Genomic analysis revealed that the three sequenced isolates were two Bacillus species and one bacteria belonging to the Exiguobacterium genus; these three isolates possess genes in the pathway of PHA degradation and were able to grow on plates where the only carbon source was PHB. In addition, the two Bacillus isolates, have genes involved in the dissimilatory reduction of sulphate, thus they have the potential to use sulphate as final electron acceptor.
The goal of the project was to isolate at least three different bacteria from the marine environment that are able to degrade a specific kind of bioplastics, called Polyhydroxyalkanoate... (More)
Isolation and genomic characterization of three PHA degrading bacteria from the marine environment.
Genomic analysis revealed that the three sequenced isolates were two Bacillus species and one bacteria belonging to the Exiguobacterium genus; these three isolates possess genes in the pathway of PHA degradation and were able to grow on plates where the only carbon source was PHB. In addition, the two Bacillus isolates, have genes involved in the dissimilatory reduction of sulphate, thus they have the potential to use sulphate as final electron acceptor.
The goal of the project was to isolate at least three different bacteria from the marine environment that are able to degrade a specific kind of bioplastics, called Polyhydroxyalkanoate (PHA).
In the field station, of Texas A&M University Corpus Christi, in the Laguna Madre, Texas, PHA pellets were deployed at the sediment-water interface and they were left there for about three months. In these three months, environmental bacteria were able to attach to the pellets and a bacterial biofilm was formed on the pellets. After three months the pellets were collected from the environment and they were taken into the lab.
The in vitro part of the project thus began. In the lab we prepared two different liquid media to isolate the wanted bacteria. From a previous study from Dr. Turner’s lab it was noticed that on the PHA pellets there’s an accumulation of sulphate reducing bacteria (SRB), much higher compared to their accumulation on plastic (PET) pellets and ceramic pellets. For this reason a procedure that involved a step in a liquid medium specific for the isolation of SRBs and a step in a liquid medium where the only carbon source was PHB (polyhydroxy-butyrate, which is part of PHAs) was implemented. This procedure would thus favor the growth of bacteria that can reduce sulphate and also degrade PHB.
Eleven different isolates were obtained with this method and for three of those, DNA was extracted and sent to sequencing. We then studied the growth rates of these bacteria at different conditions, like different temperatures (15, 30 and 37°C) and under aerobic or anaerobic conditions. Also, the isolates could grow on MSM-PHB plates in aerobic conditions but not in anaerobic conditions, interestingly enough since all the isolation steps were performed under anaerobic conditions.
The final part of the project consisted in assembling the sequenced genomes and defining to which species the isolates belonged to. We assembled each genome with three different programs, called Velvet, Spades and MaSuRCA because we wanted to see which one produced the best possible assembly. After comparing the assemblies, using a program called Quast, we saw that for all three of the isolates the best assembly was given by Velvet.
For the genome identification we then used two methods, first we performed a pair-wise comparison of average nucleotide identity (ANI); this tool is able to identify possible match with known genomes and if the match is higher than 96% it means that the two compared genomes belong to bacteria from the same species. We also made a genome-scale phylogenetic analysis, and a maximum-likelihood (ML) tree was generated. Comparing these two methods the same conclusion could be drawn: one of the isolates is part of the genus called Exiguobacterium while the other two are part of the Bacillus genus.
These findings were really interesting because not lots of species in the genus Exiguobacterium are known and also few of them were actually cultured in labs. However all the known species were isolated from a big variety of environments, ranging from glaciers to hot springs. Also finding Bacillus species that are able to degrade PHA and also reduce sulphate was surprising, since no previous information was found in the literature about any Bacillus species being able to use sulphate as final electron acceptor. The predicted increase in PHA production may result in higher PHA deposition rates in coastal sediments, which coupled with the already high concentration of sulphate could result in growth conditions favoring these types of bacteria. (Less)
Please use this url to cite or link to this publication:
author
Elli, Giulia LU
supervisor
organization
course
KBTM01 20191
year
type
H2 - Master's Degree (Two Years)
subject
keywords
PHA, degradation of PHA, marine bacteria, sulphate reducing bacteria, SRB, SRM, biotechnology, bioteknik
language
English
id
8992898
date added to LUP
2019-09-12 14:02:51
date last changed
2019-09-12 14:02:51
@misc{8992898,
  abstract     = {{The broader goal of this project was to investigate what kind of effects the accumulation of polyhydroxyalkanoates (PHA) in the marine environment might have on the microbial world. Bacteria were isolated from biofilms fouling PHA pellets placed in the sediment-water interface; the goal was to isolate PHA degraders that were also sulphate reducers. Eleven strains were isolated and for three of them genome sequencing and genomic analysis were performed. Two of them (named SRB1LM and SRB3LM) turned out to be Bacillus strains and genes involved both in PHA degradation pathways and dissimilatory sulphate reduction pathway were detected in their genomes. These two strains also grew on agar plates where the only carbon source present was PHB (poly-(3-hydroxybutyrate)); they are facultative anaerobes and their growth at 15, 30 and 37°C was studied in laboratory conditions. In a complex medium, they both reached a higher growth rate (µmax) at 37°C; in particular, SRB1LM had the highest µ at 37°C in anaerobic conditions (0.407 h-1), while SRB3LM had the highest µ at 37°C in aerobic conditions (0.849 h-1). The third bacterial genome sequenced belonged to the Exiguobacterium genus, whose known species and strains have been isolated from a variety of environments. This isolate (named SRB7LM) can also grow on PHB agar plates, it is a facultative anaerobe but does not have genes of the dissimilatory pathway of sulphate reduction. SRB7LM had a similar µ at the three tested temperatures in anaerobic conditions (0.2 h-1), which were higher compared to the ones in aerobic conditions. 
The two Bacillus strains could be interesting for the future since the increase in PHA production will cause a higher accumulation in the marine environment; this, coupled with the already high concentration of sulphate, could arise favoring conditions for their growth.}},
  author       = {{Elli, Giulia}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Isolation and genomic characterization of three PHA degrading bacteria from the marine environment}},
  year         = {{2019}},
}