10.1109/ULTSYM.2009.

0359

Non-destructive evaluation of the 18th century ship

wreck Vrouw Maria
Ari Salmi, Joona Eskelinen, Marko Peura and Edward
Hggstrm

Kari Steffen and Leone Montonen

University of Helsinki
Department of Applied Chemistry and Microbiology
Helsinki, Finland

University of Helsinki, Department of Physics

Division of Materials Science
Helsinki, Finland
AbstractWe analyze the current condition of pine and oak
samples recovered from a sunken, 240 years old Dutch merchant
ship wreck, the Vrouw Maria, with ultrasound and other nondestructive methods. To evaluate its physical condition mechanical properties- ultrasonic through-transmission sound
velocity measurements at 0,3-4 MHz were performed along the
radial and longitudinal wood fiber directions. Presence of large
discontinuities (e.g. holes created by ship worms) within the
samples were probed using 2 MHz ultrasonic pulse-echo
measurements. DNA-based methods (community DNA
extraction, amplification (PCR) of SSU rDNA, cloning,
restriction fraction length polymorphism (RFLP), sequencing)
probed presence of microbes, particularly fungi, in the samples.
X-ray diffraction and X-ray fluorescence measurements were
performed with a synchrotron to determine the extent of
degradation at the nanometer-level in the wood cell walls and
changes in the elemental composition of the samples. The
ultrasonic measurements detected a layered structure in the pine
sample featuring a thin (1-4 mm) mechanically degraded layer on
top of almost intact wood (60 % decay in the stiffness modulus
compared to the intact part). For the oak sample, a 49% stiffness
modulus reduction was detected compared to a freshly felled
reference sample. DNA-analysis detected the presence of soft-rot
fungi in the degraded layer and in the underlying compact wood
(to 2 cm depth). Fungal DNA was more abundant below the
heavily degraded layer indicating that fungal degradation had
moved deeper towards the non-degraded parts. X-ray diffraction
and fluorescence indicated presence of Fe throughout the sample
and heavy degradation of the crystalline cellulose in the degraded
layer. The results offer insight into the condition of the entire
ship wreck, and therefore support decision making regarding
possible lifting of the ship.

to preserve the ship is ongoing. It has been suggested to

preserve the ship in situ [2], but recently the possibility to lift
the entire wreck and preserve it in a museum has been
discussed [3]. Information about the physical condition and
about possible ongoing chemical reactions within the
shipwreck provides support for making this decision.
Another shipwreck, the Vasa (sunken 1628 in the
Stockholm harbor), was lifted before such studies were made,
and only post lifting, problems with internal formation of
sulphuric acid were noticed [4]. The wreck Vrouw Maria,
offers an opportunity to detect and prevent such deterioration
while the ship is still in situ. Ongoing research combines an
array of non-destructive techniques to evaluate the current
condition of the shipwreck. The goal of the research is to
predict possible future degradation.
II.

METHODS

A. Samples
A 50 cm long pine (Pinus genus, Fig. 1) and a 10 cm long
oak sample were salvaged from the ship wreck and stored in
sealed containers immersed in water originating from the
wreck site. Small cubic samples were cut out of the larger
pieces for quantitative ultrasonic stiffness modulus
determination from both sample types. In addition, a small
sample was cut out of the degraded layer (Fig. 1) in the pine
samples.

Keywords -ultrasound, xray, dna, archaeology, structural

integrity

I.

INTRODUCTION

Wooden shipwrecks exist around the world. Evaluating the

condition of such cultural heritage lying on the bottom of
oceans is essential for archaeologists. The decisions on whether
to lift objects or to leave them in situ require knowledge about
both their current and projected future condition. In
Scandinavia, the preservation of the Dutch shipwreck Vrouw
Maria [1], sunken in 1771 to a depth of 41 m in the Finnish
archipelago, has been a hot topic; the controversy around how

978-1-4244-4390-1/09/$25.00 2009 IEEE

1479

1 cm

Figure 1. (left) Schematic diagram of the sample. Samples taken for the
different experiments are marked. Layer thickness (blue = biofilm, light
brown = degraded layer) exaggarated for clarity. (right) Close-up of the cut
surface of the pine sample exhibiting a degraded layer (marked with an arrow)

2009 IEEE International Ultrasonics Symposium Proceedings

For molecular biology studies, three different samples #1,

#3, and #4, of the soft outmost layer of wood and biofilm were
collected by scraping it off with sterile scalpels and were
transferred into sterile 1.5 ml Eppendorf tubes. One sample,
#2, from the underlying harder wood beneath sample #1 was
taken, by boring and cutting into the wood to a depth of 2 cm.
The samples were frozen and stored at -20 C until processing.
For the X-ray diffraction studies, several tangential and radial
2 (thickness) x 15x 45 mm3 cubes were cut out of both the
intact and the degraded part of the pine sample.
B. Ultrasonic condition estimation
5-cycle ultrasonic longitudinal 10 VPP tone bursts at 300
kHz (longitudinal wood direction, oak), 1.25, and 4 MHz
(radial and tangential wood direction, pine) were transmitted
through the samples and then amplified 60 dB (Panametrics
5660C). The receiving and transmitting transducers were
water-coupled to the sample. An Agilent 33120A signal
generator produced the transmitted pulses, which were received
and digitized with a digital oscilloscope (WaveRunner). The
time-of-flight (TOF) of the signals was determined from the
first discernible peak of the received signal, whereas the
thickness of the samples was determined with a micrometer
measuring the distance between the transducers in situ during
the measurement. The stiffness modulae of the samples was
calculated from the determined velocities assuming isotropy by
the standard formula [5]. The sample density was determined
by measuring the volume of the cubes by a digital caliper, and
weighing the samples using a Presica 6000D scale. The
stiffness values were verified with a commercial indenter.
Pulse-echo imaging with an ultrasonic phased array setup
(MicroPulse 5PA, Peak NDT, UK) was conducted to show the
degraded layer structure and to find possible structural
discrepancies inside the sample. A 128 element (1mm pitch)
2MHz linear array transducer (Imasonic, France) was used in
water immersion. A 32 element linear scan was employed
through a 6-8 cm water path. The scan was focused on the
sample surface.
C. Molecular biology
DNA was extracted from 0.25 g of the samples using the
Powersoil DNA extraction kit (Mobio laboratories, Inc.
Carlsbad, CA, USA) according to the manufacturers protocol
and the 100 l DNA elutes were frozen until further use.
DNA was used as template for PCR amplification
(Eppendorf Mastercycler gradient machine, Hamburg,
Germany) of the 18S rRNA gene. Fungal primers NS1F (GTA
GTC ATA TGC TTG TCT C) and FR1R (AIC CAT TCA
ATC GGT AIT) which cover the gene, 1650 bp were used.
PCR reactions were done under the following conditions, 100
l (buffer 10x 10 l, MgCl2 7 l, dNTP (10M each) 2 l,
NS1F (20 pmol/l) 2 l, FR1R (20 pmol/l) 2 l, H2O 76, 5
l, Red Hot polymerase 0.5 l (2.5U) The reaction conditions
were initial denaturation at 95C for 5 min, 40 cycles of 94C

1480

for 1 min, 47C for 1 min, 72C for 2 min and a final 72C for
15 min.
The size of the
PCR products was checked by
electrophoresis in 1.5 % agorose gels stained with ethidium
bromide in 1x SB buffer and visualised by UV light, or in the
case when products were to be cloned, in 1.5 % agorose gels
stained with SybrSafe in 1x TAE buffer and visualised on a
Dark Reader.
Gel bands of the correct size were excised and DNA extracted
and used for cloning using the pGEM-T Easy kit (Promega,
Madison, Wisconsin, USA). Positive clones were selected by
blue-white screening and 40 clones of each library were
screened and tested for inserts of the correct size, by
performing a PCR with the original primers.
Restriction fragment length polymorphism analysis
(RFLP) was performed on clones containing inserts of the
correct size with restriction enzymes Fast Digest XapI and
Fast Digest HinP1I. The segments were separated on 1%
agarose with 0.5% Synergel gels and the resulting RFLP
patterns were grouped visually. Colonies with distinct patterns
were selected for sequencing, 24 clones from each library
were sequenced.
Chromatograms were inspected and sequences manually
edited in 4Peaks (A. Griekspoor and Tom Groothuis,
mekentosj.com).The sequences were compared to all available
in the GenBank+EMBL+DDBJ+PDB databases using Blastn
2.2.19. Sequences were aligned using the SINA web aligner in
the SILVA package, imported into ARB where the alignment
was manually corrected. Neighbour-joining, maximum
parsimony and maximum likelihood phylogenetic trees were
constructed using the ARB package.
D. X-ray diffraction and fluorescence
X-ray diffraction (XRD) and fluorescence (XRF)
measurements were performed at the European Synchrotron
Radiation Facility (ESRF), beamline ID 18F. The incident xray beam of 14.4 keV photon energy was collimated to a 2 * 7
m2 beam. A 25 mm distance within the samples was linescanned using a step size of 100 200 m. The
instrumentation of the beamline allowed both the XRD and
XRF measurements to be made at the same sample position.
The XRD patterns were recorded using a CCD detector with
2048*2048 pixels of 80 m size (MAR Research, Germany).
The exposure time was 1 sec for each pattern. XRF spectra
were measured using a Si(Li) detector with 30 mm2 active
area (Gresham, UK). To have sufficient counting statistics for
the XRF spectra, the measurement time was 5-20 seconds per
spectrum.
The XRD spectra were analyzed using Matlab (The
MathWorks Inc., Natick, MA, USA). The analysis
concentrated on the diffraction pattern of crystalline cellulose,
from which the reflection 200 (See Fig. 4) [6] was studied.

2009 IEEE International Ultrasonics Symposium Proceedings

The full width at half maximum (FWHM) of the reflection

was used to calculate the width of the cellulose crystallites
relying on the Scherrer formula [7].

has degraded 49% for the oak parts of the ship wreck
compared to the stiffness of fresh wood.
Figures 2 and 3 feature two ultrasonic scans of the sample,
one perpendicular to the degraded surface layer (radial wood
direction) and one perpendicular to a sawed, intact surface
(longitudinal wood direction). The duration of the surface
echo differs in these two figures, showing a more
inhomogeneous structure in the degraded surface scan. The
acoustic impedance contrast between the degraded and the
intact layer produces a reflection from the interface. This is
seen as a rise in the reflection amplitude and permits
estimating the approximate thickness of the degraded layer.
Fungal sequences were retrieved from the Vrouw Maria
samples, Table I. Based on sequence and RFLP data the
Ascomycetes sequences dominated the clone libraries.
Acremonium-like sequences were most common, constituting
a majority in the wood samples. Cultured relatives contain a
Mn(II)-oxidising enzyme with laccase activity.

Figure 2. Ultrasonic (2 MHz) scan image from the degraded layer surface.
The interface between the degraded and the intact layer is visible. This allows
estimating the thickness of the degraded layer.

Figure 3. Ultrasonic (2 MHz) scan image from a sawed, intact surface

showing a more clear interface compared to the degraded surface.

The XRF spectra were analyzed using the programs PyMCA

(ESRF, France) 4.3 and Matlab. The emphasis was on
analyzing the spectra for occurrence of trace elements from S
to Br and to deduce the homogeneity of the trace element
content in the imaged positions of the samples.
III.

Clones related to Nais inornata, a fungus found only in

submerged wood, were only obtained from the deeper wood
sample. N. inornata produces soft rot in wood and exhibits
laccase activity. The upper wood sample yielded Lulwoana sp.
sequences. These marine lignicolous fungi use submerged
hardwood and softwood as substrate. A Cryomyces related
sequence was obtained was from a clone from one of the
biofilm samples. The known Cryomyces, are Antarctic slow
growing cryptoendolithic fungi, highly resistant to cold, UVlight, freeze-thaw cycles and a good tolerance of NaCl up to
7.5%. Arthrobotys oligospora is a nematode trapping fungus
and an A. oligospora related sequence was obtained from the
upper wood sample.
Chytridiales-related fungal sequences were retrived from
one of the biofilm samples. These fungi are important
degraders of cellulose, chitin and keratin.
TABLE I.

PERCENTAGE OF DIFFERENT FUNGAL SEQUENCES IN 4 CLONE

LIBRARIES CONSTRUCTED FROM THE WATERLOGGED PINE SAMPLE
Location in the sample

RESULTS

The ultrasonically determined stiffness modulae along the

radial direction for the intact part of the pine sample were
3.40.1 / 3.30.1 GPa, and 1.40.12 / 1.30.3 GPa for the
degraded part using 4 MHz / 1.25 MHz frequencies,
respectively. Thus, the ultrasonic though-transmission
measurements indicated a 60% decay in the stiffness modulus
in the degraded layer. The ultrasonically measured modulus
values along the tangential wood direction for the intact /
degraded part (3.70.3 / 1.80.3 GPa, 5110 % decay) were
compared to the values obtained with the indenter, 6714 %
decay in the stiffness modulus in the degraded layer. For the
fresh felled oak sample, a stiffness modulus of 18.41 GPa
was ultrasonically measured along the longitudinal wood
direction. The salvaged oak sample exhibited a stiffness
modulus of 90.7 GPa, indicating that the stiffness modulus

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Genus
Acremoniumlikea
Lulwoanalikea
Nais inornatalikea
Cromycesa
Arthrobotrys
oligosporaa
Chytridiales
Non-fungal
sequences

Solid
wood (0-1
cm, S1a)

Solid wood
(1-2 cm, S2)

Biofilm
(S3)

Biofilm 2
(S4)

71%

67%

17%

41%

6%

23%

2%

3%

17%

20%

10%

83%

40%
a. Ascomycetes
b. Lower fungi

2009 IEEE International Ultrasonics Symposium Proceedings

The detected layer-like degradation pattern corresponds to

that reported in a qualitative study on waterlogged wood [11].
This report stated that some samples showed only superficial
attack, where the outer layers were soft and decayed, while the
inner parts showed no signs of microbial attack.
XRF showed the presence of Fe and heavier elements in
both degraded and well-preserved samples, similar to the
elements found in the Vasa [4]. This causes challenges for the
preservation of the ship wreck. Quantitative analysis of the
XRF spectra to determine the trace element content in the
samples is in progress.
The results offer insight into the condition of the entire ship
wreck, and therefore support the decision making carried out
by archaeologists related to a possible lifting of the ship wreck
giving information on the future degradation in both scenarios
(ship left in situ vs. lifted).

Figure 4. X-ray diffraction patterns (left) and X-ray fluorescence spectra

(right) from the degraded layer (above) and the well-preserved part (below).
The reflection 200 of crystalline cellulose is marked on the pattern. The x-ray
fluorescence peaks, starting from 3.6 keV correspond to Ca, Ti, FeK, FeK,
CuK ZnK, CuK, ZnK, AsK and BrK,

ACKNOWLEDGMENT

The XRD patterns measured from the severely degraded

layer showed a high degree of degradation of crystalline
cellulose (Fig 4). In the well-preserved samples reflections
arising due to crystalline cellulose were observed. The width of
the cellulose crystallites in the well-preserved parts was 28
29 ( 0.2) , similar to the width of cellulose crystallites in
conifers in general [7].

The authors thank Mr. Jonne Haapalainen for his valuable

help with the indentation measurements. The Maritime
Archaeology Unit of the National Board of Antiquities in
Finland is thanked for providing the samples.

The XRF spectra showed high fluorescence intensities of Fe

in all cases (Fig. 4). High fluorescence intensities of heavier
elements than Fe were observed sporadically. The XRF spectra
of both well-preserved and degraded samples were different
compared to those from normal conifer samples [8], in which
Fe and the heavier elements are not markedly present.

[1]

IV.

DISCUSSION AND CONCLUSIONS

The softwood components of the Vrouw Maria ship wreck

were degraded in a layer-like manner; a small, ~1-4 mm thick
layer of heavily damaged material was detected and quantified
on top of nearly intact wood. The mechanical properties
featured 49% degradation in the oak parts along the
longitudinal direction matching the measured degradation of
the elastic modulus (50%) of the Vasa ship wreck [9]. The
layer formation was visible in the ultrasonic pulse-echo
measurements, indicating that an in situ measurement of the
thickness of the layer at the ship wreck could be possible.
Most fungal sequences retrieved from the Vrouw Maria
clone libraries belonged to soft-rot causing marine
Ascomycetes [10]; this matches the detected stiffness
degradation. Acremonium-like, Lulwoana-like and Nais
inornata-like fungi may possess ligninolytic in addition to the
commonly occurring cellulolytic properties of the
Ascomycetes. The biofilm covering the ship wreck consists of
bacteria, fungi, plankton and degraded wood. The Chytridiales
and zooplankton present in the film may degrade cellulose, as
was seen in the X-ray results.

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2009 IEEE International Ultrasonics Symposium Proceedings