Professional Documents
Culture Documents
(Microbial physiology
and Metabolism)
BTY538
Leptospirillum
ferooxidans
SUBMITTED BY:
SHASHI SHARMA
(RP8003B15)
Msc.Microbiology(2403)
Reason for selection of Leptospirillum
ferooxidans
Leptospirillum ferooxidans
Heap bioleaching is presently the most successful technology
for copper extraction from low-grade sulfide ores. Metabolic
behaviors and adaptation mechanisms of microbial communities
to this system remain unknown. A combination of molecular
approaches has been used to determine the key
microorganisms in the Escondida Sulphide Heap. This work
reports the global gene expression in industrial bioleaching
samples by using a specific DNA microarray of Leptospirillum
ferrooxidans to better understand the metabolic functions
displayed by Leptospirillum spp. iron oxidizers as mineral
leaching proceeds. Quantitative real-time PCR analysis showed
low cell numbers of Leptospirillum species during the first part
of the irrigation period, however, it became the most abundant
and active in the second part of the cycle. Transcriptome
analysis by DNA microarray could provide specifics
transcription patterns in the different stages of the industrial
process. We studied the gene expression from bioleaching heap
samples with variable Leptospirillum cell numbers, operation
times and with different chemical and physical conditions. The
results showed induction of genes involved in energetic
metabolism, electron transport, ribosomal proteins, DNA repair
and response to heavy metals when Leptospirillum species were
most abundant and active. We suggest that up- and down-
regulation of specifics genes could be used as indicators of
physiological responses to operational conditions in the
bioleaching systems.
Contents
1 Classification
1.1 Higher order taxa:
1.2 Species:
Industrial importance
Genome Structure
4 Cell Structure and
Metabolism
5 Ecology
6 References
Classification
Species:
Leptospirillum ferriphilum, Leptospirillum ferrooxidans,
Leptospirillum sp
Industrial importance
Leptospirillum is a genus of iron-oxidizing bacteria which play
an important role in industrial bioleaching (the conversion of
metals to a soluble form) and biooxidation (the extraction of
metals). They are obligate aerobes (require
oxygengas).Leptospirilla have been found to be the primary
iron-oxidizers in industrial continuous-flow biooxidation tanks.
The reason for this bacteria's domination in biooxidation is
most likely the fact that the high ferric-ferrous iron ratio
inhibits all but species of Leptospirillum. Leptospirilla are also
important contributors to the acid mine drainage process;
these combine to make Leptospirilla more important players in
the acid mine processes than previously thought.
Genome Structure
Studies on the genetics of bacterial components of acid mining
systems have revealed Leptospirillum's prominence in several
areas of the process, leading to new revelations about the
bacteria's role in the industry. Sequencing of certain isolates'
16S rRNA has demonstrated the existence of new species of
Leptospirilla. There have also been several plasmid sequences
done to Leptospirillum ferrooxidans. The first one, called
Leptospirillum ferrooxidans plasmid p49879. 1, was 28878 bp
long, and the second, called Leptospirillum ferrooxidans plasmid
p49879. 2, was 28012 bp long.
Leaching results
Bioleaching of pyrite by L. ferriphilum YTW315 The
bioleaching results of pyrite by YTW315 at 40 ℃ are shown in
Fig.7. In the whole process, total iron extraction continuously
increased. From 10 to 25 d, leaching rate of iron increased
significantly and finally reached 95.31% after 30 d. Almost no
soluble iron was detected in un-inoculated controls all the time.
The data indicate that L. ferriphilum YTW315 has great
capacity of leaching pyrite, and this result is consistent with
previous reports[. ZHANG Rui-yong, et al/Trans. Nonferrous
Met. Soc. China 20(2010) 135−141 140
Ecology
Acid stream draining from Iron Mountain, CA. Image from
NOAA.
DISCUSSION
Thiobacilli dominate the microflora in the samples of the
Ronneburg heaps, although the cell counts of COT seemed to
be higher in many samples. Nevertheless, because lithotrophic
bacteria including thiobacilli were measured by the most-
probable number technique, the real cell count was
considerably underestimated . This results from the growth
mode of lithotrophs. Organisms growing in a biofilm cannot be
sep-
arated into single cells. This holds for leaching bacteria as well.
Hallmann et al. demonstrated attached cells of L. ferrooxidans
on ore particles.
T. ferrooxidans was regularly detected in the samples,whereas
L. ferrooxidans only rarely occurred. Similar results were
described by Groudev and Groudeva, Goodman et al. , and Sand
et al. for Bulgarian, Australian, and Romanian leach biotopes,
respectively. Obviously, the growth requirements of L.
ferrooxidans (temperature above 208C and availability of
water are not met. However, with the substrate pyrite, L.
ferrooxidans could be detected in several samples. This raises
the possibility that there is a population of L. ferrooxidans
which is not culturable with soluble ferrous iron. Further
experiments are planned to address this question.The use of
microcalorimetry enabled us to determine that at the
Ronneburg site the leach activity of T. ferrooxidans was 10
times lower than elsewhere. Obviously, T. ferrooxidans is
considerably inhibited at this site. The reason for this
inhibition remains to be elucidated. The loam layer covering
heap A seems not to be decisive. The alkaline pH value, due to
the carbonaceous gangue material, and/or the considerable
degradation of the ore, resulting in a muddy consistency of the
top layers, may be responsible.
Moderately acidophilic thiobacilli were detected more often
than T. ferrooxidans at the heap A sites, where the pH values
were mainly around 7. The question of their substrate
arises,because small numbers of T. ferrooxidans and T.
thiooxidans were found at these sites. According to the
equation of the indirect leaching mechanism , sulfur would be
available. However, a few sulfur producers cannot produce
enough sulphur to feed many moderately acidophilic sulfur-
consuming thiobacilli. Furthermore, the leach mechanism
requires acidic pH values. However, preliminary data indicate
that the leach equations must be revised . Thiosulfate and
ferrous iron are proposed to be intermediates of the oxidation
of pyrite by ferric iron . There is evidence that thiosulfate
instead of sulphur is the primary product of microbial pyrite
leaching under acidic conditions . This finding suggests the
existence of an altered sulfur chemistry based on sulfane
monosulfonic acids. Thiosulfate undergoes a sequence of
chemical reactions which finally may give rise to the easily
detectable and slowly biodegradable sulfur. Furthermore,
thiosulfate, trithionate, and tetrathionate have previously
been detected in the course of the oxidation of pyritic mine
spoil material and during chemical oxidation of pyrite at pH
values between 6 and 9 . Thiosulfate is known to be a good
substrate for the moderately acidophilic thiobacilli. These
bacteria are able to FIG. 2. Occurrence of anaerobes as a
function of depth. (a) Uncovered part of heap A; (b) covered
part of heap A. Symbols: ‚, lithotrophic nitrate-reducing
bacteria; ‚ ƒ, organotrophic nitrate-reducing bacteria; F,
sulfate-reducing bacte- ria; p,Fe31-reducing bacteria; h,Mn41-
reducing bacteria.
FIG. 3. Variation of microbial activity in different habitats. ,
Ronneburg
(50 samples, r 5 0.93); – – –, Cartagena (66 samples, r 5 0.96);
·········, Ilba (82 samples, r 5 0.97); ——, percolators (22 samples,
r 5 0.96). r, correlation coefficient. The habitats are uranium
mine waste heaps near Ronneburg (this work); waste heaps
from Cu, Zn, and Pb mining near Cartagena, Spain ; the Mina
Ilba in Romania (in situ leach experiment ; and laboratory
experiments in percolators .
VOL. 61, 1995 MICROBIAL DIVERSITY IN URANIUM MINE
WASTE HEAPS 2933grow if pyrite is chemically oxidized at
neutral pH values (1).This may explain, why moderately
acidophilic thiobacilli occur in large cell numbers in samples
from the covered part of heap A, where neutral pH values are
found. They were detected in small cell numbers in heap G,
where pH values around 4 were measured. However, a
significant correlation between the occurrence of bacteria and
the pH could not be demonstrated. This probably resulted
because the ore material contains more than 90% shale.
Suspending the samples during processing resulted in an
increased availability of carbonaceous compounds. This caused
a change of the original pH in microniches of the ore to an
overall (mixed) value.The physical parameters temperature and
gas composition are important for the diversity of the
microflora. As a consequence of the high temperature, a
thermoacidophilic bacterium resembling the leach bacteria
Sulfolobus/Acidianus spp.could be detected at a hot sampling
site. The gas composition inside the heaps changed with depth.
The oxygen concentration decreased, and the carbon dioxide
concentration increased. Corresponding to the decrease in
oxygen concentration, the number of T. ferrooxidans cells
decreased. Obviously, at a depth of 1.5 to 2 m, the oxygen
concentration became limiting for the growth of T.
ferrooxidans. Concomitantly, the carbon dioxide concentration
increased (mainly as a result of the dissolution of the
carbonaceous gangue material). Similar data were
demonstrated for Australian mine waste heaps and for other
leach biotopes . Consequently, the cell counts of T.
ferrooxidans remained constant up to a depth of5min
Australian mine waste heaps under microaerophilic
conditions .Generally, it is accepted that lithotrophic bacteria
feed acidophilic COT by the excretion of organic compounds .
The cell counts of the latter correlated with the cell
counts of T. ferrooxidans, obviously in agreement with this
hypothesis. The decrease in the cell counts of the acidophilic
COT at the uncovered sites may be explained by the depth
dependent decrease in the T. ferrooxidans cell counts. For all
other microorganisms, a similar decrease with depth was noted
irrespective of aerobic or anaerobic metabolism. The main
nutrient source for the microorganisms is the pyrite oxidation
by T. ferrooxidans. If this process does not function, nutrients
for the other types of microorganisms soon become exhausted.
Atmospheric deposit by rain or dust seems to be negligible.
Anaerobically growing bacteria may be important members of
the biocoenosis in leach dumps because they are able to reduce
insoluble metal compounds to a soluble and, hence, mobile
species. The reductive solubilization of ferric iron and
manganese oxides and hydroxides may result in a release of
coprecipitated heavy metal ions . Among the anaerobic
bacteria, the nitrate-reducing ones are the most important.
Nitrate was regularly detectable in the samples, ensuring a
constant supply for these bacteria. The nitrate might result
from the activity of nitrifiers present in the heaps, although
the general importance of nitrifying bacteria is obviously low.
They occurred only at the surface of the heaps and were
present in
small numbers. Sulfate-reducing bacteria were detected in
samples from the surface, although aerobic conditions exist.
This might be explained by the finding that some species are
able to use oxygen as an electron acceptor .A large microbial
diversity exists in mine waste heaps. Rehabilitatio measures
must be scrutinized for their effect on the microbial
biocoenosis and the leach activity, if they are to be
successfully realized. The microcalorimetric leach activity
measurements constitute a notable advancement for this
task.The effect of measures becomes detectable within a
reasonable time span. To take the right action, the leach
process must be thoroughly understood and further research
is needed to elucidate the sulfur chemistry in leach heaps.
Conclusions
1) A new strain L. ferriphilum YTW315 is successfully isolated
by overlay agar solid medium. This medium is more efficient
for isolation of L. ferriphilum compared with other methods. In
this medium, it is presumed that Acidiphilium sp. PJH
facilitates the formation of L. ferriphilum colonies.
2) The cells of YTW315 are Gram-negative and are motile,
vibrio- or spiral-shaped, with a diameter 0.3−0.5 μm and a
length of 1.1−2.6 μm. It has a guanine plus cytosine (G+C)
content of 58.1% and exhibits similarity of 99.8% to L.
ferriphilum strain Fairview and ATCC 49881.
3) Physiological investigation indicates that L.ferriphilum
YTW315 is a strict chemolithoautotroph, metabolizing ferrous
iron and pyrite. The optimal temperature of L. ferriphilum
YTW315 is 40 ℃ and the optimal pH is 1.6 for growth. The
strain has high tolerance to As(Ⅴ) with the concentration 50
mmol/L.
4) The leaching rates of iron and copper by L. ferriphilum
YTW315 are 95.31% and 44.56% after 30 d, respectively, and
the results reveal L. ferriphilum YTW315 is very useful in
bioleaching of metal sulfides ores.
References