The document summarizes three presentations on microbial eco(physio)logy: 1) Behavior of bacteria degrading pentachlorophenol (PCP) in soil ecosystems by the University of Helsinki. It found that Mycobacterium effectively degraded PCP in soil for 290 days while Flavobacterium's activity declined after 60 days. 2) Ecophysiological mechanisms of cyanobacterial blooms by the University of Amsterdam. It studied the cellular behavior and cell wall proteomics of a cyanobacterium under nitrate-N stress and found the cell wall adapted specifically to different nitrogen sources. 3) Microbial identity within microbial ecology by Wageningen Agricultural University and Helsinki University. It aimed to

1. Lessons from Microbial Eco(physio)logy:
a few examples.
1. Behaviour of bacteria degrading
PentaChloroPhenol (PCP) in soil ecosystems;
(University of Helsinki, FIN).
2. Ecophysiological mechanisms involved in
cyanobacterial bloom; (University of Amsterdam, NL).
3. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Dr.M.Briglia

2. Presentation structure
Content of each part:
 Scientific and practical questions leading to
the research work;
 Aims, Experimental approach and M&M;
 Results and Conclusions;
 Room for questions.
Dr.M.Briglia

3. Lessons from Microbial Eco(physio)logy:
few examples.
1. Behaviour of bacteria degrading
PentaChloroPhenol (PCP) in soil
ecosystems; (University of Helsinki, FIN).
2. Ecophysiological mechanisms involved in cyanobacterial bloom;
(University of Amsterdam, NL).
3. Microbial identity within microbial ecology (WAU, NL & HU, FIN).
Dr.M.Briglia

4. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Dr.M.Briglia
Place:
 University of Helsinki
 Department of General Microbiology
Participants:
 Prof. Dr. Ir. M. Salkinoja-Salonen
 Dr. M. Briglia
 Dr.Ir. P.J.M. Middeldorp, Dr. Ir. V. Kitunen, Dr.Ir.
R.Valo, and the technical supporting staff.

5. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
 Why is it relevant to carry out pilot study on
bacteria which are already known to attack and
degrade PCP?
 Because scientific evidence on their degrading
performance and behaviour under field conditions
would allow a more efficient and controllable use of
the chosen bacterium besides gaining more
knowledge.
Dr.M.Briglia

6. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Experimental Aims:
 To understand why externally introduced
xenobiotic degrading bacteria seldom perform
well under field conditions;
 To improve biodegradation of PCP in soils by
using more effectively degrading bacteria;
 To explore sustainable soil bioremediation
possibilities.
Dr.M.Briglia

7. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Experimental approach:
 Study of the bacterial survival capacity in different
types of soils;
 Explore correlation/predictability between lab-data
knowledge versus natural ecosystems;
 Investigate the effect of contamination and
inoculums levels, and natural amendments on
bacterial viability and activity.
Dr.M.Briglia

8. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
M & M:
 Inocula of PCP-degrading bacteria: -
Flavobacterium
and +
Rhodococcus/(Mycobacterium) PCP-1;
 Soil types: pristine sandy and peaty soils (Kettula
farm, Suomusjärvi, FIN);
 Natural amendments: Distillery Waste and Wood
Chips;
 Polyurethane (PUR) foam 90/16 type containing
active carbon as bacterial carrier;
 Pentachlorophenol crystals 97% purity and
uniformly 14
C-labelled PCP 96% radiochemical
purity, 10.57 mCi mmol-1
.
Dr.M.Briglia

9. Electron micrographs of thin sections of PUR-foam containing active carbon
before and after colonization with M. chlorophenolicus PCP-1. a) sterile, b)
after colonization by a pure culture, … next …
Dr.M.Briglia

10. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Chemical structure of PCP
Dr.M.Briglia

11. Experimental set up:
Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Dr.M.Briglia

12. Behaviour of bacteria degrading PCP in soil ecosystems:
Experimental observations:
Dr.M.briglia

13. Behaviour of bacteria degrading PCP in soil ecosystems:
Experimental observations:
Dr.M.briglia

14. … c) a similar PUR-foam pads as in b), but sampled after 290 days exposure to
PCP contaminated soil w/o amendments, and d) sampled as in c) but in PCP
contaminated soil added with DW. The arrows indicate presence of cells.
Dr.M.Briglia

15. Behaviour of bacteria degrading PCP in soil ecosystems:
Experimental observations:
Dr.M.Briglia

16. Behaviour of bacteria degrading PCP in soil ecosystems:
Experimental observations:
Dr.M.Briglia

17. Behaviour of bacteria degrading PCP in soil ecosystems:
Experimental observations:
Dr.M.Briglia

18. Behaviour of bacteria degrading PCP in soil ecosystems ...
Results:
Dr.M.Briglia
•Mycobacterium chl. PCP-1 remained viable and
kept its PCP-degrading activity for up to 290 days
regardless the different simulated field conditions i.e.
± PCP contaminant and ± natural amendments;
•Flavobacterium lost its viability as well as its
degrading activity within 60 days under the same
simulated field conditions;
•PCP was mineralized up to 97%;
•The addition of natural amendments did not
improve the bioremoval of PCP in the studied soils.

19. Behaviour of bacteria degrading PCP in soil ecosystems ...
Conclusions:
Dr.M.Briglia
•Field PCP biodegradation can be done
effectively by using PUR-immobilized
Mycobacterium chl. PCP-1 inoculum at cell
density of ca. 108
cells/g.s.d.wt.;
•Addition of natural amendments is not needed
to improve the bacterial PCP-degrading activity;
•Laboratory bacterial performance is not always
equal to field performance.

20. Behaviour of bacteria degrading PentaChloroPhenol
(PCP) in soil ecosystems; (University of Helsinki, FIN).
Dr.M.Briglia
Questions

21. Lessons from Microbial Eco(physio)logy:
a few examples.
1. Behaviour of bacteria degrading PentaChloroPhenol (PCP) in
soil ecosystems; (University of Helsinki, FIN).
2. Ecophysiological mechanisms involved
in cyanobacterial bloom; (University of
Amsterdam, NL).
3. Microbial identity within microbial ecology (WAU, NL & HU, FIN).
Dr.M.Briglia

22. Eco-physiological mechanims involved in
cyanobacterial bloom
Place: University of Amsterdam
Laboratory of Microbiology
Participants: Prof. Dr. L. Mur
Dr. H.J.M. Matthijs
Dr. M. Briglia
Ir. J. Balke
Dr.M.Briglia

23. Eco-physiological mechanims involved in
nitrogen-N stress cyanobacterial bloom
Why
Nutrient stress?
(nitrate-N) Environmental factors
Intervention
Prevention
Cyanobacterial
bloom
Dr.M.Briglia
How
Cell behaviour
and
Proteomics of the cell wall

24. Eco-physiological mechanims involved in
cyanobacterial bloom
 Why is it important to study nitrate-N stress?
 Because:
*N is necessary for growth and metabolism of
the cell;
*nitrate-N is one of the inorganic sources for N;
*prior to its reduction it can be used for different
cell purposes:
Dr.M.Briglia

25. *... it can be used prior to its reduction for
different cell purposes:
Assimilatory = NO3
-
NO2
- NH4
+ Gln
Glu
respiration
dissimilation
Dissimilatory =
Dr.M.Briglia

26. 1. Elucidate cyanobacterial behaviour under
nitrate-N stress;
2. Determine whether the cyanobacterial cell
wall responds specifically to nitrate stress;
3. Develop molecular tools to monitor bloom-
warning signals (multiprobe array: identity +
activity monitoring).
Experimental Aims:
Dr.M.Briglia
Eco-physiological mechanims involved in
cyanobacterial bloom

27. Eco-physiological mechanims involved in
cyanobacterial bloom
Dr.M.Briglia
Experimental approach:
1- Growth under nitrate-N stress
conditions; (Cellular behaviour)
2- Molecular structure changes at the
cell wall level; (Proteomics of the
cell wall).

28. * Batch culture system (rich and
depleted nitrate conditions);
* Continuous culture system
(nitrate inputs 0.5 and 0.05 mM,
d=0.015, NH4
+
input 0.05 mM);
* Cyanobacterium strain model
Synechocistys PCC 6803.
M & M:
Cellular behaviour
Proteomics of the cell wall
Approach:
Dr.M.Briglia

29. Effect of nitrate-N stress on the behaviour of
Synechocystis cells
 Under nitrate depletion Synechocystis cells
undergo a quick loss of pigments
(bleaching);
 They keep dividing though at almost
undetectable level;
Experimental observations (batch culture):
Dr.M.Briglia

30. 0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0 2 4 6 8 10 12 14
Time (day)
Absorbance(750nm)
control
NH4+ limitation
NO3- limitation
light limitation
light limitation
Fig. 1
modulation phase
modulation phase
Dr.M.Briglia
Effect of nitrate-N stress on the behaviour of
Synechocystis cells
Experimental observations (continuous culture):

31. Effect of nitrate-N stress on the behaviour of
Synechocystis cells
 Low nitrate input slows down the growth;
 The type of nitrogen source influences the
growth of Synechocystis PCC 6803;
 The modulation phase of nitrate limited cells
is shorter than that of ammonium limited
cells.
Results:
Dr.M.Briglia

32.  Elucidate cyanobacterial behaviour under nitrate-N stress;
 Determine whether the cyanobacterial
cell wall responds specifically to
nitrate-N stress;
 Develop molecular tools to monitor bloom-warning
signal/s (multiprobe array: identity + activity).
Experimental Aims:
Cellular behaviour
Proteomics of the cell wall
Dr.M.Briglia
Approach:

33. Effect of nitrate-N stress on strain PCC 6803 cell wall:
study of the protein pattern.
* Isolation of the cell wall fraction
(by floatation ultracentrifugation on discon-
tinuous sucrose density gradient);
* Analysis of the cell wall fraction
(by SDS-PAGE and polypeptide molecular
weight determination);
M & M:
Dr.M.Briglia

34. 1. Cell disruption by shearing forces (bead beating);
2. Preparation of discontinuous sucrose density gradient;
10%
30%
45%
48%
55%
90%
Before
centrifuging
After
centrifuging
Cytoplasmic
membrane
Cell wall
Dr.M.Briglia
Isolation of the cell wall fraction
M & M:

35. Analysis of the cell wall protein pattern
Experimental observations:
1) SDS-PAGE of the cell wall protein pattern of PCC 6803 cells submitted
to rich (+) and depleted (-) nitrate treatment.
+ + +- - -
66,2 Kb
45 Kb
2.2ųg 1.8ųg 1.4ųg 1.2ųg 0.7ųg 0.6ųg
31 Kb
21,5 Kb
97,4 Kb
116,2 Kb
200 Kb
Dr.M.Briglia

36. 2) SDS-PAGE of the cell wall protein pattern of PCC
6803 cells submitted to sufficient (+), limited (-)
nitrate and ammonium (NH) treatment.
200 Kb
97,4 Kb
116,2 Kb
66,2 Kb
45 Kb
31 Kb
21,5 Kb
+N -N -NH
7ųl 10ųl 10ųl 10ųl7ųl 7ųl15ųl 15ųl 15ųl
Dr.M.Briglia
Analysis of the cell wall protein pattern
Experimental observations:

37. Effect of nitrate-N stress on strain PCC 6803 cell wall:
study of the protein pattern.
* Depletion of nitrate-N induces synthesis of new
polypetides in the cell wall of strain PCC 6803 as
shown by SDS-PAGE analysis;
* Induction of the synthesis of these proteins
occurs already at low nitrate-N concentration (0,05
mM);
* Low concentration (0,05 mM) of ammonium-N
induces also synthesis of new protein.
Results:
Dr.M.Briglia

38. Molecular ecophysiology of strain PCC
6803/cyanobacteria under nitrate-N stress.
Conclusions:
1) Indeed strain PCC 6803 responds
specifically to the stress of different Nitrogen
sources.
2) In strain PCC 6803 nutrient stress (N)
induces a specific adaptation of the cell wall
rather than a non-specific increase of its
permeability.
Dr.M.Briglia

39. 1. Elucidate cyanobacterial behaviour under
nitrate-N stress;
2. Determine whether the cyanobacterial cell
wall responds specifically to nitrate stress;
3. ... Future study: develop molecular
tools to monitor bloom-warning signals
(multiprobe array: identity + activity).
Experimental Aims:
Dr.M.Briglia
Eco-physiological mechanims involved in cyanobacterial
bloom

40. Eco-physiological mechanims involved in
cyanobacterial bloom
Dr.M.Briglia
Questions

41. Lessons from Microbial Eco(physio)logy:
a few examples.
1. Behaviour of bacteria degrading PentaChloroPhenol (PCP) in
soil ecosystems; (University of Helsinki, FIN).
2. Ecophysiological mechanisms involved in cyanobacterial bloom;
(University of Amsterdam, NL).
3. Microbial identity within microbial
ecology (WAU, NL & HU, FIN).
Dr.M.Briglia

42. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Dr.M.Briglia
Places: Wageningen Agricultural University,
Laboratory of Microbiology
&
Helsinki University,
Department of General Microbiology
Participants: Prof. Dr. Ir. W. De Vos (NL)
Prof. Dr. Ir. M. Salkinoja-Salonen (FIN)
Dr.Ir. G. Schraa (NL)
Dr. M.Sc. M. Briglia (FIN-IT-NL)
and the technical supporting staff (FIN-NL)

43. Microbial identity within microbial ecology (WAU,
NL & HU, FIN).
Dr.M.Briglia
Why study microbial identity?
Because it helps to gather information needed to
place the studied microbe somewhere in the
culture collection and mainly to give it a name so
that it is no more an unknown “identity” and when
you need it you can find it back;

44. Microbial identity within microbial ecology (WAU,
NL & HU, FIN).
Dr.M.Briglia
When to study microbial indentity?
If:
 you need another paper to finish your Ph.D.
 or you do not have much work to do (lacking
brilliant ideas period), and
 by accident you have just isolated a bacterium
that resembles anything else but itself,
 etc. etc.

45. Microbial identity within microbial ecology (WAU,
NL & HU, FIN).
Dr.M.Briglia
Experimental aims:
 To create another original scientific article;
 To define the identity of a bacterium that
appeared to do a nasty job that other known
bacteria did not want to do, and that especially
seems to have a strange look;
 To check whether it might have ancestors that
are harbouring similar behaviour.

46. Microbial identity within microbial ecology (WAU,
NL & HU, FIN).
Dr.M.Briglia
Experimental approaches:
 Determine the xenobiotic degrading and
physiological properties, morphology, size, and
ultrastructure that are relevant to its taxonomical
classification;
 Define the sequence of 16S rDNA and perform
phylogenetic inference;
 Design DNA probe to specifically detect the microbial
target;
 Develop protocol for the isolation of DNA in the
ecosystem under study.

47. Microbial identity within microbial ecology (WAU,
NL & HU, FIN).
Dr.M.Briglia
M & M:
 One PCP-degrading bacterium known as
Rhodococcus chlorophenolicus strain PCP-1;
 One 2,4,6-TCP and DCP-degrading newly isolated
bacterium;
 Pristine and contaminated soil samples.

48. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Experimental observation:

49. Microbial identity within
microbial ecology
(WAU, NL & HU, FIN).
Experimental observations:
1. The genus specific helix of the
16S rRNA of strain PCP-1
strongly resembles that of the
genus Mycobacterium;
2. The inferred phylogenetic
relationship to all of the
species belonging to the genus
Mycobacterium examined
indicated similarity values
greater than 95%;

50. Microbial identity within
microbial ecology
(WAU, NL & HU, FIN).
Experimental observations:
1. Along the 16SrRNA
molecule three
nucleotides streches
could be identified for
designing a specific
detection probe;

51. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Dr.M.Briglia
Experimental observations:
1. The designed probes were tested for specific
detection of strain PCP-1 in inoculated soils
and showed specificity to the target A), B) and
C);
2. The sensitivity of the detection method was
tested at different inoculums densities and
detection was possible down to 3x102
cells p.g.
soil.

52. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Results:
1. The taxonomical data and genetic
determination based on the sequence analysis
of 16S rDNA gene showed that Rhodococcus
chlorophenolicus strain PCP-1 belongs no
longer to the genus Rhodococcus but to the
genus Mycobacterium.
2. The nucleic acid probe allowed specific
detection of strain PCP-1 in soil and enhanced
detection of PCP-1 down to 3x102
cells p.g. soil
d.wt.

53. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).

54. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Electron micrographs of 2,4,6-TCP and DCP-degrading
newly isolated bacterium;

55. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).

56. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).

57. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Results:
The taxonomical data, genetic determination
based on the sequence analysis of 16S
rDNA gene, and phylogenetic inference
allowed the classification of the 2,4,6-TCP
degrading bacterium as Rhodococcus
percolatus sp. nov. strain MBS1.

58. Microbial identity within microbial ecology
(WAU, NL & HU, FIN).
Conclusions:
 Molecular genetics and phylogenetic inference
ensure an accurate microbial classification;
 Nucleotidic probes allow a more sensitive, specific,
and quicker microbial identification in environmental
samples;
 Thorough taxonomic and phylogenetic
characterization of microbes can support
understanding of microbial behavior in natural
ecosystems.
 ... And much more ...

59. Microbial identity whithin microbial ecology
Recent developments:
Metagenomic Analyses: past and future trends,
Minireview by:
C. Simon and R. Daniel, AEM, Feb. 2011, p. 1153-1161.
... A few extracts:
Metagenomic bypasses the need for isolation or cultivation of microorganisms. Metagenomic
approaches based on direct isolation of nucleic acids from the environmental samples
have proven to be powerful tools for comparing and exploring the ecology and metabolic
profiling of complex environmental microbial communities, as well as for identifying novel
biomolecules by use of libraries constructed from isolated nucleic acids ...
Metatranscriptomics provides information on the actual metabolic activity ... Metatranscriptomic
studies of microbial assemblages in situ are rare. ....
Metaproteomic analysis of mixed microbial communities ia a new emerging research area which
aims at assessing the immediate catalytic potential of a microbial community. ...