Isolation, characterization and possible biocontrol application of Bdellovibrionaceae (BD) isolated from NZ sources : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) at Massey University
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Date
2008
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Massey University
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Abstract
Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the
world's smallest living hunter they prey on other Gram-negative bacteria giving them potential
as biological control agents. Prior to this study, however, there were no reports of BD in New
Zealand. The overall aim of this research was to isolate BD from New Zealand sources,
characterise them and investigate their potential role as a biological control agent. The history,
characteristics, life cycle and mechanism of predation of this organism are reviewed and the
possibility of the industrial applications of BD, are discussed.
In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites
around New Zealand. Thirteen isolates were characterised using proven characterisation
techniques including general, microscopic and molecular techniques. It was found that the
isolates were taxonomically identical or very closely related to each other and belong to the
genus Bacteriovorax.
The predation pattern of BD isolates was examined against a group of Gram negative bacteria in
solid and liquid media. The predation patterns and efficiencies of the different BD isolates were
similar, which confirms that the BD isolates are closely related, are selective in their predation,
and prey on some Gram-negative bacteria but not all.
The rapid loss of culture viability of BD is well known, but no studies have been reported to date
on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense
suspensions at different temperatures without host bacteria was investigated and it was observed
that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging
from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and
37°C after 13 to 16 days.
The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium
phosphoreum were examined to select the best isolate for in vitro application. All of the isolates
tested had considerable reduction effect against P. phosphoreum. Some isolates were more
effective than others, despite their taxonomic similarity to each other. The isolate OT2 was
selected for further studies based on these results.
The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage
bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions
of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of
incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to
8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and
predator concentrations and nutrient concentration. Prey concentrations were observed to be the
most vital factor in predation and the most favourable predation conditions were at a prey
concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration
of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds
of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and
3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of
BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative
spoilage bacteria, the normal condition observed in saltwater fish. There has been very
little research in this field and the results of these trials suggest further investigation into the
effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only
one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in
liquid medium, the evidence of these findings must be restricted to these particular conditions.
Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic
organisms in solid medium are warranted.
The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A
significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended
through extension of the lag phase of growth of the prey bacteria and a reduction in total
numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf
life was extended. However, at 10°C there was no reduction in prey organisms, which suggested
that the strain of BD used is ineffective at refrigeration temperatures.
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Keywords
Bdellovibrio bacteriovorus, Photobacterium phosphoreum, Fishery products, Biocontrol, New Zealand, Biotechnology