79 •Es imposible elaborar escalas de preferencias de los consumidores de espacios públicos por cuanto estos
5.2.4 Propiedades que deben revestir los indicadores
The culture collection at AgResearch covered a broad diversity of rumen methanogens
representing various genera. Due to the large number of cultures that had been accumulating
for decades, some were stored without being formally identified. This project was initiated
from the necessity to inspect the viability of these cultures and to assign phylogenetic
identities to the strains. In the parallel phylogenetic trees constructed based on 16S rRNA and
mcrA gene sequence similarities, the majority of strains grouped within the genus
Methanobrevibacter. The 16S rRNA gene sequence similarity of the strain D5 to its closest relative (Methanobrevibacter millerae) was slightly less than the 98% cut-off threshold for
species differentiation, suggesting the potential phylogenetic novelty of this strain.
So far, only 3 species of the genus Methanosphaera have been isolated in pure cultures.
Methanosphaera stadtmanae, isolated from human faeces, was the first species of the genus Methanosphaera that had the full-genome sequenced (Fricke et al., 2006). Genome analysis of Methanosphaera stadtmanae revealed that the organism has specially adapted to colonize
the human gut (Fricke et al., 2006). Methanosphaera cuniculi was isolated from rectal
samples of rabbits (Biavati et al., 1988). Strain ISO3-F5 has been very recently isolated from
sheep and is the first true rumen isolate of the genus Methanosphaera (Jeyamalar, 2010).
Genome sequencing of ISO3-F5 is currently under way to reveal its adaptive strategies to
enable the survival of the strain in the rumen environment.
In this study, a pure culture of strain A4 was obtained from a culture derived from the
foregut of a Tammar Wallaby (Macropus eugenii). According to the phylogenetic analysis,
66
A
B
C
D
E
F
Figure 3.3 Fluorescence and phase-contrast microscopic images of methanogen isolates. Fluorescent (A,C,E) and phase-contrast (B,D,F) microscopic images of isolates 229/11, AbM4 and G16.
229/11 229/11
AbM4 AbM4
67
G
H
I
J
K
L
Figure 3.3 continued. Fluorescent (G,I,K) and phase-contrast (H,J,L) microscopic images of isolates Alpaca, M1 and D5.
Alpaca Alpaca
M1 M1
68
M
N
O
P
Q
R
Figure 3.3 continued. Fluorescent (M,O,Q) and phase-contrast (N,P,R) microscopic images of isolates R4C, YCM1 and BRM9.
R4C R4C
YCM1 YCM1
69
S
T
U
V
W
X
Figure 3.3 continued. Fluorescent (S,U,W) and phase-contrast (T,V,X) microscopic images of isolates MCB-3, ISO3-F5 and A4.
MCB-3 MCB-3
ISO3-F5 ISO3-F5
70
Table 3.4 Morphological descriptions of methanogen isolates used in this study. Isolate Cell shape Cell length
(µ m)
Cell width (µ m)
Specific characteristics
229/4 rod 2.0-3.0 1.0-1.3 Sometimes occur in chains
229/5 rod 2.0-3.0 1.0-1.3
1.0-1.3
229/11 rod 2.0-3.0
229/14 rod 2.0-3.0 1.0-1.3
229/15 rod 2.0-3.0 1.0-1.3
AbM4 rod 2.7-6.0 1.3-2.0 Transparent spaces at cell
ends
AbM4 rod 2.7-6.0 1.3-2.0
AbM23 rod 2.7-6.0 1.3-2.0
AbM25 pseudosarcina Irregular irregular
AL10 pseudosarcina Irregular irregular Irregular cell clumps
CM2 pseudosarcina Irregular irregular
CM3 pseudosarcina Irregular irregular
R4C rod 2.0-3.3 1.7-2.0 Isolated from Wallaby
Alpaca coccobacillus 1.3-2.7 1.3-2.0
SM9 coccobacillus 2.0-3.3 1.5-2.5
AS9/11/19 rod 0.7-0.9 0.5-0.8 Occur in chains
G16 coccobacillus 2.0-3.3 2.0-2.7
D5 rod 2.7-4.0 2.0-2.7 Derived from H6 A4 coccus 2.7-3.7 2.7-3.7 Isolated from wallaby
MCB-3 coccus 3.3-4.0 3.3-4.0 Human isolate ISO3-F5 coccus 2.7-3.3 2.7-3.3 BRM9 Ccooked long rod > 4.0 0.7-1.3 Filamentous
YCM1 long rod > 4.0 0.7-1.3 Filamentous
71 Methanosphaera cuniculi. Wallabies, along with kangaroos, belong to the marsupial family Macropodidae. Native to Australia, marsupial animals evolved in geographically separated
regions from the ruminant animals. Strictly speaking, macropod marsupials do not possess
the rumen, but their foregut carries out similar functions to the rumen by harbouring a
complex microbiome in which bacteria, archaea, fungi and protozoa interact to breakdown
plant materials. For an unknown reason, the methane production from marsupial animals is
relatively low compared to the ruminants (Dellow et al., 1988, Kempton et al., 1976, Engelhardt et al., 1978). It is still too early to describe the contribution that the members of the genus Methanosphaera have on the overall production of ruminant methane, as there are
so few isolates available for study. Nevertheless, methanogens of the genus Methanosphaera
are one of the predominant inhabitants of the rumen of New Zealand livestock (Jeyamalar et
al., 20011). A comparative genome analysis of Methanosphaera spp. isolated from the rumen and the foregut of marsupials may reveal the genomic variations that cause the
production of varying amounts of methane from these species. Genomic variations between
these species may suggest (1) that the contribution of the genus Methanosphaera towards the
overall production of ruminant methane may be relatively small; (2) methanogens inhabiting
the foregut of marsupials are too small in numbers to produce a significant amount of
methane; and (3) the marsupial foregut microbial ecosystem is not optimized for
methanogenesis.
With the phylogenetic identities of the methanogens at hand, the next step will be to
characterize the phenotypic properties of the strains of interest. A majority of the methanogen
strains used in this study still has not been formally described. The complete description of a
microbial species is carried out through the characterizations of the substrate and growth
72 taxonomic and morphological properties, and tolerance against chemicals inducing cell lysis
such as sodium dodecyl sulphate (SDS), lysozymes, proteases and NaCl solutions. It was not
the aim of this work to complete a full characteristization; rather, growth characteristics that
75