October, 2013
Newsletter
Issue 3
Welcome
to the third
newsletter for the MOSSCLONE
project which is financed by the EU
Commission’s Seventh Framework
Programme. The aim of this project
is to develop and implement a method
to control the air quality by using
a devitalized moss clone as passive
contaminant sensor.
This newsletter introduces the
midterm meeting of the MOSSCLONE
project which encompasses the hard
work of more than 30 experts from
11 organisations representing 5 EU
countries (Spain, Italy, France, Ireland
and Germany).
We are happy to report about
some great work that is being done on
this EU organisations. The tasks are
being carried out at the appropriate time
and with the expected results, including
critical tasks like moss cultivation and
the analytical optimization of PAHs. We
can say that Europe is halfway to have
an important new tool for measuring
any kind of air pollution anywhere.
The MOSSCLONE Newsletter
is published twice a year by the Project
Manager. If you wish to be included or
taken off the distribution list, please
contact him (carlosbrais.carballeira@
usc.es).
Edited by Carlos Carballeira
What is MOSSCLONE?
Two European Directives on ambient air quality assessment (96/62/EC of 27
September 1996 and 2008/50/EC of May 2008) obliges the Member States
to deliver periodically precise information about the air quality and the
related health within their territories to ensure that the European population
is aware of it.
For compliance to both Directives, States usually use monitoring stations,
but these stations are only useful when macro-pollutants are assessed in
agglomerations. For measurement of other pollutants included in the
Directives there are technical difficulties and their analysis on air is too
expensive. As a consequence there is a lack of representative data through
Europe. In addition, data from automatic devices are accurate but too limited
in number of pollutants and to describe spatio-temporal trends of pollutants.
Due to the limitations of traditional methods, bio-monitoring is an adequate
alternative to acquire data about the levels of pollutants that affect European
citizens and makes it possible to evaluate the state of environmental
parameters inf luenced by synergistic effects of different pollutants.
Among the available bio-monitors, terrestrial mosses are especially adequate
for air quality assessment due to their high efficiency in loading both
particulate and gaseous determinants of organic, inorganic, and radioactive
pollutants.
However, there are some problems that can arise when using mosses for
the current moss-bag technique: the absence of well-suited moss species
living in urban, extra-urban, and even indoor reference environments; the
bags are prepared from mosses naturally grown in unpolluted areas, so its
availability and the natural variability on moss elemental composition could
vary depending on natural and anthropogenic causes.
The solution to avoid these problems is to cultivate in the laboratory
a moss clone to always have homogeneous material with the same
initial concentrations to prepare the bags. In this way, a high degree of
standardization would be reached and would allow a comparison of the
exposed mosses in the same way by means of Enrichment Factor or Net
Enrichment.
An additional, but highly relevant problem that usually affects the use of biomonitors is the lack of standardized protocols and methodologies. The lack
of such protocols hampers comparison of the results obtained in different
studies, and sometimes limits the conclusions that can be reached. The
MOSSCLONE approach would overcome all these issues, thereby improving
data quality and reproducibility, and therefore usability of environmental
data collected throughout Europe.
1
Project Goals
The main MOSSCLONE objectives are:
1. Selection of moss species on the basis of their use as bio-monitor,
their distribution and their physico-chemical characteristics.
2. Creating a pilot bioreactor for the cultivation and the selection
of the most suitable species.
3. Characterization of the selected moss clone.
4. Scaling up moss clone cultivation.
5. Design and methodological standardization of the moss-bag
technique.
6. Moss-bags validation versus current state-of-the-art methods
for air pollution monitoring.
7. Perform an initial validation of its usefulness for the detection of
atmospheric small scale pollution focus.
RECENT MOSSCLONE EVENTS
Midterm meeting at Freiburg (Germany)
This work was carried out
as part of WP5 (Exploitation and
dissemination) of which the main
objectives were: exchange of final
and preliminary results from finished
and developing tasks; stimulate crossWP communication; to strengthen
the collaboration between WPs; to
discuss scientific bottlenecks and to
learn how to prepare the financial
reporting.
The full consortium meeting took
place on the 26th and 27th of September
(2013) at the FRIAS building, located
in Freiburg (Germany). The meeting
was focused on the progress of all
WPs. A ll the partners, advisory
board members, the Project Officer
and several stakeholders were invited
to attend the meeting.
Partners met each other, presented the
development of activities and work
packages and clarified the doubts they
had about some tasks of the project.
ALU-FR (University of Freiburg) was
the partner in charge of this meeting.
The meeting ended as a productive
and friendly event where the finished
and current activites were discussed
and the expectations of all attendees
were fullfilled. The meeting was one
day and a half of presentations and
discussions, and one day of fieldtrip.
The schedule of the agenda was
almost adjusted to perfection (see
page 4).
2
3
Meeting Agenda
The midterm meeting was one day and a half of presentations and discussions and one
day of fieldtrip. The first day was focused on showing the results obtained in finished
and currently developed activities and discuss this results and the proceedings of next
activities. Once again, the schedule of the agenda was adjusted to perfection.
26th September
Morning (09:15 – 12:30)
9:00-9:15 Registration
9:15-9:30 Welcome address
9:30- 10:00 WP2 : General presentation and summary of results from WP leaders
meeting (AMRA)
10:30-11:15 Results (or preliminary results) obtained from the clone (I)
Molecular characterization (AMRA)
Multi-elemental characterization (UDC,AMRA)
11:15-11:45 Coffee break
11:45-12:05 Results (or preliminary results) obtained from the clone (II)
Physical-chemical characterization (CNRS)
12:05-12:30 WP3: Overview of standardization assays (Biovia)
12:30-14:00 Lunch Break
Afternoon (14:00-17:30)
14:00-14:20 WP3: Standardization assays. Preliminary analytical results (TELabs)
14:20-15:30 Critical Task: Cultivation and scaling up of the clone (ALU-FR, Biovia)
Cultivation method and scaling up
Biovia workplan (moss bag and large-scale production)
Discussion
15:30-16:00 Coffee break
16:00-17:30 WP4: Detectors workplan (Orion)
Task 4.1. Standard bag vs bulk deposition
Task 4.2. Cover bags vs particle samplers
Task 4.3. Cover bags with diffusers vs gaseous samplers
Discussion
20:00
Consortium Dinner at Dattler restaurant
27th September
Morning (09:30 – 12:30)
09:30-9:50 Exploitation and dissemination: Past and Future (ALU-FR)
09:50-10:20 How to prepare the periodic report (USC)
10:20- 10:50 Coffee break
10:50-11:10 WP1 Project management- State of art (Tasks, Reports, Time,…) (USC)
11:10-12:30 General Discussion and Conclusions
12:30-14:00 Lunch Break
Afternoon (14:00-17:30)
14:00-16:00 Optional: A visit to the Reski Lab
17:00-18:30 Historix City Tour
19:00 Dinner at the Martins Bräu in Freiburg
4
Summary of main
achivements of the meeting
• Reinforce team building
• Prepare the Financial reporting
• Ensure proper communication between WPs
• Presentation of results from the various tasks
• Planning of WP4
• Optimization of the critical activities that are described on the DOW
to the current state of the project
WP2 Cultivation and characterization
Critical tasks
Analytical optimization
Several studies have been done during this activity: i) State of the art in the analysis of PAHs in moss.The majority of articles used Hypnum sp. and the Soxhlet technique), ii) Look for drawbacks of the previous
method.- Less coextraction of interfering substances, perform the evaporation of 12 samples simultaneously,
better results for most volatile PAHs, less consumption of sorbents and solvent and a less laborious method, iii)
Optimization and Improvements of the method.- Less consumption of sorbents and solvents, cost reduction,
less laborious, shortening of the analysis time, iv) Study of alternative analytical procedures.
The findings obtained in the different subtasks allowed us to develop an optimized and validated procedure for the analysis of PAHs in moss using microwave assisted extraction. The proposed method improves
considerably the initial procedure according to the principles of green chemistry: Less consumption of toxic
solvents, automation, less laborious and time consuming. The mspd initial experiments seems to provide great
results. The initial selected moss species to this tests was Pseudoscleropodium purum. However, the finally
selected species to be cloned is Sphagnum palustre so the results must be checked.
Cultivation and up-scaling of the clone
In vitro cultures of mosses selected within task 2.1 (literature review for species selection) were obtained (Sphagnum palustre, Rhynchostegium murale, Brachythecium rutabulum, Hylocomium splendens, Pseudoscleropodium purum and Hypnum cupressiforme). However, S. palustre was selected according to the results
obtained on the physical-chemical characterization for the species selection (task 2.2). For this reason we have
finally decide to continue our cultivation studies just with this species.
We have established an axenic culture of Sphagnum p. by following this steps: 1) Sterilization from
spores in capsules, 2) Germination of spores, 3) Isolation of single protonema, 4) Cultivation of independent
clones.
The cultivation in different mediums and growth experiments for medium optimization have been done
using: i) Solid medium, ii) Hydroponic gametophore cultivation on sieves and iii) Liquid medium in Erlenmeyer flasks.
We have also established a dry weight measurement method and the up-scaling of the moss clone in
bubble flasks and bioreactors. Moreover, we have found that disruption with an Ultraturrax is lethal for S. palustre so we have looked for alternative disruption techniques. Finally, selected species adjusts itself the pH of the
culture (nature and bioreactor).
5
WP3 Tool development
Pictures from sampling at an industrial area (up left), a street with vehicle
traffic (down left) and the device for the exposure of samples at different
heights (right).
Exposure map
This map shows the regions where the samples were exposed
(Galicia (Spain), Campania (Italy), Vienna and Lower Austria
(Austria).
6
The samples required for the
standardization of the exposition
method have already been
exposed and processed for their
analysis. This experiment was
developed without almost any
incident. Only 7 of about one
thousand samples disappeared
from sampling points, which
may be mainly caused by
strong winds and robbery.
Samples are already in TE
Labs for the analysis of their
metal content (Aluminium,
Arsenic, Barium, Beryllium,
Cadmium, Chromium, Cobalt,
Copper, Iron, Mercury, Nickel,
Palladium, Lead, Platinum,
Rhodium, Selenium, Strontium,
Tin, Vanadium and Zinc).
Differences between native and
cloned moss
Native
Moss
Clone
Average
weight per
shoot
Leaf
Leaf section
Length
Hyalocysts
Chlorocysts
larger and
shorter,
with robust
thickenings
and numerous
pores
narrow
diameter
longer, thin
walled, with
1-2 pores
larger
diameter
Native
14 mg
cucullate
wideness
ratio
about 2:1
Clone
3.5 mg
not
cucullate
very
long and
flexuose
An analysis of polymorfisms
in DNA regions showed no
differences between different
lines of the clone while there
were differences between
these lines and native moss.
Analysis of Moss Samples
Moss samples from standardization experiments were to be analysed for metal content.
For this purpose, microwave digestion was done prior to analysis. Then, samples were
analysed for metal content using Inductively Coupled Plasma-Mass Spectroscopy (ICPMS) in a dedicated ICP-MS.
A moss reference material was used to carry out the necessary method development
necessary to validate the procedure decided upon and to optimise recoveries of desired
metals. A new cleaning procedure after each digestion run was also performed.
Elements of interest were Al, As, Ba, Be, Cd, Cr, Co, Cu, Fe, Hg, Ni, Pd, Pb, Pt, Rh, Se,
Sr, Sn, V and Zn.
Currently, 70 samples are being analysed weekly. All analysis and results will be complete
by mid December (2013).
7
Physical-chemistry Characterization
The adsorption of heavy metals (Cu2+, Cd2+, Ni2+, Pb2+ and Zn2+) on 4 typical moss
species (Hypnum sp., Sphagnum sp., P. purum and B. rutabulum) was studies in order to
be used for environmental monitoring purposes. The surface acid-base titration performed
demonstrated that Sphagnum sp. is the most efficient adsorbent as it has the maximal number
of proton-binding sites on the surface, although the relative difference from other mosses is
within 20% despite the species and geographic differences.
The pKa computed for all the moss species suggested the presence of 5 major functional
groups: phosphodiester, carboxyl, phosphoryl, amine and polyphenols. The quantitative
comparison of metal adsorption with other common natural organic and inorganic materials
demonstrates that mosses are among the most efficient natural adsorbents of heavy metals.
The isotopic fractionation of Zn during adsorption experiments indicated that is very weak
which means that Zn can be used as true environmental monitor in case of pollution tracing (no
artifact during uptake).
FORTHCOMING EVENTS
The next WP meeting (2º) will take place the Friday 29 th in Santiago de Compostela,
hosted by Biovía.
Tasks
Recent
Cultivation of moss
clone and Analytical
optimization
(see page 5)
Current
Multi-element
Physical-chemical
Molecular
Characterization
The full characterization
of the clone is being
done. These activities are
almost finished.
Scaling up to large
scale clone production
These activities are also
almost finished. It only
remains to optimize, a bit
more, moss production
using bioreactors.
Sphagnum palustre
8
Upcoming
Clone moss bag
preparation
We have already started
clone production using
bioreactors. Once we
have enough quantity of
cloned moss we will start
preparing the moss bags
for the next task (WP4).
WP4 activities
The beginning of this
tasks depend on the
availability of cloned
moss. During the last
meeting we have decided
to adjust the number
of sampling times
and replicas making
moss production to be
affordable enough for the
proper development of
the Project.
MOSSCLONE Dissemination
During these 18 months the MOSSCLONE project has been disseminated through different pathways:
Internet
Website and intranet: There
is a public access to our website
through the URL www.
mossclone.eu. Moreover, there
is an intranet only accessible
for mossclone members and
where some information related
to the project is uploaded.
Wikipedia article of the
MOSSCLONE
project
(English
and
German
languages): Sites were viewed
324/162 times in the last 90
days.
Facebook group has 50 members
TV
Newspapers
Our project coordinator has
been interviewed by two
Spanish national tv channels:
La Sexta Noticias and
Noticias Cuatro.
The international TV channel
Euronews has made a video
about Mossclone within a
documental series (Futuris).
Here you may find the latest
news about the leading
scientific & technological
research projects in Europe.
Universities:
Freiburg
(Germany)
and
Santiago
de Compostela universities
(Spain).
ht t p://w w w.euronews.
com/2013/06/03/biotechnologyto-fight-air-pollution/
RNE:
The
coordinator
of mossclone project was
interviewed by the public
national Spanish radio.
National newspapers:
Razón (Spain).
Sampling
Twitter account has 33 followers
Radio
Regional
newspapers:
Mossclone was mentioned
two times in La voz de Galicia
(Spain) and Faro de Vigo
(Spain) and one time in El
Correo Gallego (Spain) and
Badische Zeitung (Germany).
Samples
were
exposed
together with a banner in order
to disseminate the project
physically in the places where
the experiment took place (see
picture on the right).
Calendar
Meet our partners in the following forthcoming events:
• To the Next 40 Years of Sustainable Global Aquaculture from 3rd-7th
November (2013) in Las Palmas de Gran Canaria
• Kyoto University Symposiums, Japan: 13th-14 December (2013)
About us
Find out more on our website,
Find Me on
Facebook
www.mossclone.eu
Follow US on
Twitter
facebook.com/MOSSCLONE
@MOSSCLONE
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