Bioremediation by means of plants and genetically modified bacteria
Summary and conclusions
 | Colonisation studies of alfalfa (Medicago sativa) by the bacterial strain Pseudomonas fluorescens F113rifpcb indicate that the strain can colonise this plant at a level of 105 cfu/g to 106 cfu/g root (fresh weight), and will constitute up to 10% of the total aerobic bacterial rhizosphere population. |
| Colonisation of alfalfa roots using beads (alginate encapsulated Pseudomonas fluorescens F113rifpcb) was found to be the most efficient way of introducing the inoculant into non-sterile soil microcosms. Different encapsulation formulations have been shown to optimise the release of the genetically modified microorganisms to soil over time. |
| Left and right distal regions of the bph cassette were sequenced, and DNA sequence information allowed the design of PCR primer pairs that can be used to amplify a unique amplicon, indicative of this DNA module, which can be used to track the recombinant insert in Pseudomonas fluorescens F113rifpcb. A method was developed using real time PCR methods for the detection and quantification of strain F113rifpcb and its recombinant genes. |
| A similar quantitative assay was developed for strain lacZY, based on a unique sequence in the junction between the inserted cassette and the wildtype chromosome. |
| A major objective of the project was to place the bph degradation genes under the control of exudate/environment inducible promoters. A model plasmid to assess an iron regulated promoter from Pseudomonas sp. strain M114 was constructed and evaluated. |
| Pseudomonas fluorescens F113 derivatives have been constructed with re-regulated bph pathways. Constructs with pNODD4 and pEX promoters located in the 5' region of the bph cassette have been evaluated for growth on biphenyl and gene expression in vitro. These strains have also been evaluated for colonisation of alfalfa roots in non-sterile soil microcosms. |
| A goal of the project was to develop inoculants with limited persistence (contained strains). A regulatory gene of secondary metabolites was targeted as a key gene required for the long term survival of Pseudomonas in soil. |
| Pex fusions to this regulatory gene have been constructed and tested, showing the feasibility of a biological containment system based on root exudate-induced expression of gacA. |
| A specific carrier is found suitable for application of strain Pseudomonas fluorescens F113 on cuttings as well as on seeds. |
| A protocol for easy surface sterilisation of willow was developed, as is the hydroponic sterile culture system of this tree species. This gives basis for more precise measurements and comparison studies of different bacteria used for rhizoremediation in combination with this promising plant species. |
| Pseudomonas fluorescens F113 is able to colonise and establish in the rhizosphere and bulk soil of willow. Over several months, the abundance of Pseudomonas fluorescens F113 remained higher in the willow rhizosphere than in the bulk soil. Killing the plants with a herbicide showed that the strain was able to survive on the decaying roots and re-establish on new willow roots emerging in the same soil. |
| A genetically modified strain of Pseudomonas fluorescens F113 harbouring the bph-genes had a potential to degrade biphenyl and some PCB congeners to CO2. Probably, the low degradation rate was due to the fact that no extra C-source was added to the medium, allowing the degradation to be cometabolic. |
| We were unable to detect improved degradation of 4-PCB in the soil as an effect of the inoculated Pseudomonas fluorescens F113 bacteria with bph-genes. One reason for this could be a low activity of the inoculated strain in the rhizosphere. As the bph-genes are expressed constitutively, the expression is decreased when strain F113 was not growing actively. The soil analyses clearly showed that 4-PCB disappearance was due to uptake by the willow roots. This was verified by extraction of the willow plants, as 4-PCB was recovered mainly from the shoots. |
| Reporter constructs of Pseudomonas fluorescens F113 with the stable variant of gfp were used as an indicator of the localisation of strain F113 at the root of alfalfa. CLSM studies showed the inoculated strain to be present along the entire roots of alfalfa. The strain colonised the rhizoplane, often in the intercellular caves between plant cells, the mucigel, root tip and root hairs. No effect of adding 4-PCB to the soil was found on the colonisation patterns. No toxic or selective effect was found of not having or having the degradation genes. This is consistent with the low activity of the bacteria located at the rhizoplane. |
| Reporter constructs with the unstable variants of gfps (gfpAGA and gfpAAV) driven by the ribosomal promoter were shown to be reporters of the gene expression of introduced cells in the rhizosphere, in that the expression was strongly dependent upon the growth rate. The CLSM studies showed that only bacteria located at the root tip or at sites for lateral root emergence expressed fluorescence from the two unstable variants of gfps. This indicate that only cells at the root tip or at lateral root emergence sites are able to maintain a certain activity based upon the leakage of exudates, and that cells located here have a higher activity than cells located elsewhere at the root. However, the missing expression from unstable gfps does not necessarily imply starvation or no activity, as the promoter can be active at levels below gfp detection. |
| The pm promoter was shown to be inducible by chlorobenzoates, which are degradation products from the degradation of PCB in Pseudomonas fluorescens F113rifpcb. Pure culture experiments showed that the induction and expression of gfp start within a few minutes, and that the concentration needed for gfp expression and fluorescence at single cell level is low. This together makes F113Pm::gfp constructs suitable reporters for the degradation of 3-PCB in the rhizosphere of alfalfa. CLSM studies showed that Pseudomonas fluorescens F113rifpcb was able to degrade 3-PCB in the rhizosphere of alfalfa. However, these studies showed that even though the introduced strain colonised the entire root, only a minor fraction of the cells was degrading at a rate sufficient to be visualised in CLSM. In these present experiments, the rhizodegradation was shown to occur when using the 3-CBA inducible pm promoter and stable gfp as a marker. However, when using the pm promoter combined with unstable gfp, no induced fluorescence was seen, indicating a low degradation rate of the xenobiotic. This is supported by the fact that we showed that the addition of PCB did not lead to a higher number of culturable cells in the rhizosphere of the PCB-degrading strain. |
| Probably, the explanation for the missing measurable degradation of 4-PCB in the soil microcosms experiments is that only a small fraction of the introduced cells remained active and that the degradation rate was too slow to be measurable within the time of the experiment. Also, uptake by the willow was found to be an effective competitor for the available PCB to Pseudomonas fluorescens F113rifpcb. The conditions for a successful rhizodegradation were found to be present, as Pseudomonas fluorescens F113rifpcb colonises the rhizosphere and is able to degrade PCBs in pure culture. |