Application of a new constant high pressure incubation system (DeepIsoBUG) to obtain and characterise novel, high pressure, deep biosphere prokaryotes

Lead Research Organisation: Cardiff University
Department Name: School of Earth and Ocean Sciences

Abstract

Recently, huge numbers of bacteria kilometres deep in the Earth have been discovered. These bacteria may represent over 50% of all life on Earth. But we know almost nothing about these bacteria as we can only grow a tiny fraction of them in the laboratory for study. Deep ocean sediments contain most of these 'deep biosphere' bacteria and here pressures are enormous over 300 times that at the surface. Up till now the only way to study them was by bringing samples to the surface and trying to study them at atmospheric pressure, which might kill the vast majority of them. We have developed a system which enables bacteria to be consistently grown and studied under high pressure. This can be used with a new pressure coring and handling system so that bacteria are never depressurised which should allow deep bacteria not previously seen to be studied. We will investigate these bacteria to find out what type they are, how they survive in their extreme (high pressure, low energy and zero oxygen) environment and how they might influence processes in deep sediments and rocks which were previously thought to be geological environments devoid of life. These deep sediments can be tens of millions of years old and may contain ancient bacteria. They may even be an environment where life on Earth might have started. So understanding these bacteria will not only tell us about life on Earth it might also tell us more about how life began.
 
Description Objectives and Results
1. To use continuous high-pressure cultivation to enrich, isolate and characterise potentially unique subsurface anaerobic bacteria using the recently developed DeepIsoBUG system.
This was conducted on gas hydrate sediment samples from the Gulf of Mexico, Cascadia Margin and Indian Continental Margin, with a range of hyperbaric pressures (0.1, 14, 40, 50, 78 and 80 MPa) and media (Nutrient Broth, Nutrient Broth plus nitrate, Sulphate reducing bacterial (SRB) plus CH4, SRB plus VFA, Methanogen plus methyl compounds, Methanogen plus VFA, Metal Reducing bacterial, Acetogen). This resulted in isolation of a large collection of Bacteria representing several phyla:
Phylum Genus Isolate No. Isolated from Sediment Pressure (MPa)
Firmicutes Carnobacterium 14 Indian Continental Margin 0.1, 14, 40, 80
5 Gulf of Mexico 14, 78
2 Cascadia Margin 14
Marinilactibacillus 11 Indian Continental Margin 14, 80
2 Cascadia Margin 14
Clostridium 1 Indian Continental Margin 80
Acetobacterium 1 Indian Continental Margin 40
Gammaproteobacteria Pseudomonas 1* Indian Continental Margin 0.1
1 Cascadia Margin 0.1
Bacteroidetes novel 1 Indian Continental Margin 14
Table 1 Collection of bacteria isolated from hyperbaric DeepIsoBUG culture
The majority of isolates were facultative anaerobes with exception of the strict anaerobic Acetobacterium and Clostridium, and the strict aerobic Pseudomonas*. These isolates have been substantially physiologically characterised and publications are being prepared. Similar types of bacteria were isolated from all 3 gas hydrate sediment samples.
2. To compare bacteria obtained without depressurisation using the coupled HYACINTH-DeepIsoBUG pressurised coring and subsampling system with: (a) depressurised sampling followed by high pressure cultivation in DeepIsoBUG,(b) discontinuous pressure incubation using batch pressure vessels and
(c) 0.1 MPa incubations.
High-pressure enrichment cultivation (hydrostatic conditions) was carried out under discontinuous high-pressure cultivation using batch pressure vessels on depressurised sediment samples from the Indian Continental Margin with a range of pressures - 0.1, 14, 40 and 80 MPa and similar media to above. Several Bacteria similar to those obtained without depressurisation were isolated Carnobacterium (8), Clostridium (22), Pseudomonas (7), Bacteroidetes (1 novel) plus Halomonas (2).
3. To use molecular genetics to monitor biodiversity of enrichments (e.g. DGGE) and to guide isolation, and assist in identification of isolates (16S rRNA gene sequence analysis).
PCR-DGGE analysis of bacterial 16S rRNA genes was used to monitor all high pressure enrichments (both continuous hyperbaric and discontinuous hydrostatic culture) to determine diversity of bacteria within the enrichment and to aid subsequent bacterial isolation.
No Archaea were detected in any of the high pressure enrichments. Bacterial populations were relatively stable with time in both hyperbaric and hydrostatic cultures even after several subcultures. Similar types of dominant Bacteria (Carnobacterium, Acetobacterium and Clostridium) ocurred in both types of enrichment culture and throughout a range of pressures. However, in hydrostatic pressure there were no Carnobacterium in any of the selective media conditions other than nutrient broth. Also under hydrostatic conditions, a higher degree of bacterial diversity, including typical deep biosphere bacterial lineages (e.g. Chloroflexi, JS1 and NT-B6) were detected at high pressure (80 MPa). On subculture this diversity decreased. Full details of the hyperbaric enrichments are In Press in Environmental Microbiology.
4. To compare 16S rRNA gene sequence analysis of isolates with those in the original sample to assess how representative they are of the in situ bacterial community.
This was conducted on Indian Continental Margin hydrate sediments (77 mbsf). DNA was screened for bacterial and archaeal 16S rRNA genes and mcrA (ANME and methanogens) and dsrA (sulphate reducers) functional genes. Nested PCR products were only obtained with general bacterial 16S rRNA gene primers. PCR-DGGE analysis showed very low bacterial diversity with only 2 detected sequences from the Gram positive groups Firmicutes and Actinobacteria. However, these were not related to sequences of any of the high-pressure enrichments. Despite this, many of the sequences in the enrichments have previously been found in other subsurface sediments. For example, Carnobacterium and Clostridium-like sequences are present in a number of subsurface environments, including Nankai Trough and gas reservoirs; similar sequences to the novel Bacteroidetes and Firmicutes are present in gas hydrate sediments.
Exploitation Route High-pressure anaerobic microbiology.
Sectors Energy,Environment