Appl Microbiol Biotechnol (2009) 83:521527
DOI 10.1007/s00253-009-1969-9
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Characterisation of a new thermoalkaliphilic bacterium
for the production of high-quality hemp fibres,
Geobacillus thermoglucosidasius strain PB94A
A. G. Valladares Jurez & J. Dreyer & P. K. Gpel &
N. Koschke & D. Frank & H. Mrkl & R. Mller
Received: 18 July 2008 / Revised: 16 March 2009 / Accepted: 16 March 2009 / Published online: 31 March 2009
# Springer-Verlag 2009
Abstract Novel thermophilic and alkaliphilic bacteria for
the processing of bast fibres were isolated using hemp
pectin as substrate. The strain PB94A, which showed the
highest growth rate (=0.5/h) was identified as Geobacillus
thermoglucosidasius (DSM 21625). The strain grew opti-
mally at 60C and pH 8.5. During growth on citrus pectin,
the strain produced pectinolytic lyases, which were excret-
ed into the medium. In contrast to the commercially
available pectinase Bioprep 3000 L, the enzymes from G.
thermoglucosidasius PB94A converted pectin isolated from
hemp fibres. In addition to hemp pectin, the culture
supernatant also degraded citrus, sugar beet and apple
pectin and polygalacturonic acid. When hemp fibres were
incubated with the cell-free fermentation broth of G.
thermoglucosidasius PB94A, the fineness of the fibres
increased. The strain did not produce any cellulases, which
A. G. Valladares Jurez : J. Dreyer : D. Frank : R. Mller (*)
Institut fr Technische Biokatalyse,
Technische Universitt Hamburg-Harburg,
Denickestrae 15,
21073 Hamburg, Germany
e-mail: ru.mueller@tu-harburg.de
P. K. Gpel : H. Mrkl
Institut fr Bioprozess und Biosystemtechnik,
Technische Universitt Hamburg-Harburg,
Denickestrae 15,
21073 Hamburg, Germany
N. Koschke
Fakultt fur Mathematik,
Informatik und Naturwissenschaften. Department Biologie,
Biozentrum Klein Flottbek und Botanischer Garten,
Universitt Hamburg,
Ohnhorststrae 18,
22609 Hamburg, Germany
is important in order to avoid damaging the fibres during
incubation. Therefore, these bacteria or their enzymes can
be used to produce fine high-quality hemp fibres.
Keywords Geobacillus thermoglucosidasius . Hemp .
Pectin degradation . Fibre retting
Introduction
Bast fibre plants such as flax, hemp, nettle and ramie
produce valuable sclerenchymatous fibres, which can be
used in various products like textiles and high-quality
papers (McGovern 1983). These cellulose fibres are
arranged in bundles parallel to the longitudinal axis of the
stem and are embedded in a pectic polysaccharide network
(Vignon and Garcia-Jaldon 1996; Cronier et al. 2005). For
utilisation of the bast fibres, they have to be separated from
the woody core by field retting and mechanical processing.
To achieve a fine quality and good separation of the fibres
from the bundles, as it is required by textile industry, the
fibres need further treatment by chemical, physical or
biological processing (Dreyer et al. 2002). Chemical
processing is done on a commercial scale by boiling with
NaOH or diluted sulphuric acid. However, this process is
energy consuming, leads to environmental problems and
can damage the fibres (Brhlmann et al. 1994; Cao et al.
1992). Physical processing involves the steam explosion
process (Kessler et al. 1998) or ultrasonic techniques (von
Drach et al. 1999) but both options are not yet industrially
feasible. With water retting of hemp plants, good fibre
qualities can be achieved, but due to water pollution and
malodour problems, the technique is banned in most
western countries (van Sumere 1992). Enzymatic retting
or enzymatic separation of coarse bast fibre bundles is
522
another possible treatment. In the enzymatic process, the
pectin matrix holding the fibres is broken by pectinolytic
enzymes. Enzymatic retting has been examined particularly
for flax fibre (Akin et al. 2002; Foulk et al. 2001; Sharma et
al. 2005; Zhang et al. 2005) but so far no commercial
process has been developed. Pectin-degrading enzyme
preparations are commercially available, but most of them
are developed for the juice industry and contain a mixture
of pectinases, cellulases and other carbohydrate-cleaving
enzymes (Kashyap et al. 2001), which make them not
suitable for fibre treatment because the contaminating
cellulase activities could lead to low fibre strength and
quality. Therefore, new pectin-degrading enzymes for the
enzymatic treatment of hemp fibres from newly isolated
microorganisms are necessary. Due to the unknown
macromolecular structure of pectins from different origins
(Schols and Voragen 2003), for the degradation of hemp
pectin, it is necessary to isolate microorganisms, which are
able to degrade especially the pectin of hemp. Therefore,
hemp pectin was chosen as substrate for the isolation of
new organisms.
Only few thermophilic microorganisms capable of
growing on pectin and their pectinolytic enzymes have
been reported so far (Kluskens et al. 2003). In order to
prevent contamination in an industrial process especially
with cellulolytic microorganisms and to obtain more stable
enzymes, high temperatures are advantageous. At the
same time, an alkaline pH helps to remove the initial
breakdown products of pectin. Therefore, we screened for
microorganisms that have the ability to grow on hemp
pectin as the sole carbon source at temperatures above
60C and at pH values above 8. Such organisms have not
been isolated before. This paper describes the isolation of
such bacteria, their properties and their ability to produce
finer fibres of higher quality than those obtained by
conventional methods.
Materials and methods
Materials
Pectin from citrus peel and polygalacturonic acid sodium
salt were purchased from Sigma-Aldrich, Taufkirchen,
Germany. Apple pectin was obtained from Herbstreith &
Fox, Neuenburg, Germany. Sugar beet pectin was obtained
from Obipektin, Bischofszell, Switzerland. Hemp fibres for
pectin extraction and fibre treatment experiments were
kindly provided by Agro-Dienst, Neerstedt, Germany.
Enzyme preparation Bioprep 3000 L was kindly provided
by Novozymes Germany GmbH, Ingelheim, Germany. All
other reagents used were from commercial sources and
were of analytical grade.
Appl Microbiol Biotechnol (2009) 83:521527
Microorganisms
Geobacillus thermoglucosidasius PB94A (DSM 21625)
and Geobacillus thermodenitrificans PB1511 (DSM
21923) were isolated from bast fibre raw material using
hemp pectin as the sole carbon source.
The 16S ribosomal DNA (rDNA) sequences were
determined and submitted to the National Centre for
Biotechnology Information (NCBI).
Cultivation media
All media were autoclaved at 121C. For solid media, agar
(15 g/L) or gelrite (7 g/L) were added.
Screening was done on minimal medium with hemp
pectin as carbon source. The minimal medium contained
per litre of demineralised water: 1.95 g KH2PO4, 9.91 g
Na2HPO42H2O, 0.38 g MgSO47H2O, 2 g (NH4)2SO4 and
1 mL of trace element solution which was prepared
according to Sanio (1995). Ammonium sulphate was
autoclaved separately and added to the medium under
sterile conditions; 12.5 g hemp fibres were extracted with
1 L of minimal medium at a temperature of 65C for 1 h to
remove highly soluble substances. The liquid was discarded
and the remaining material was extracted with 1-L minimal
medium at 121C for 20 min to extract the remaining
pectin. The latter material was used as hemp pectin
medium.
Minimal medium for cultivation of the isolates was
prepared from 980-mL solution A and 10 mL of solutions
B and C. All solutions were autoclaved separately and
added aseptically. Solution A contained per litre of
demineralised water: 1 g (NH4)2SO4, 0.66 g K2HPO4,
0.54 g KH2PO4 and 5 g of citrus pectin or polygalacturonic
acid as carbon source. Solution B contained per litre of
demineralised water: 20 g MgSO4 7H2O. Solution C
contained per litre of 0.1 M HCl: 2 g CaCl2 2H2O, 1 g
FeSO47H2O, 0.5 g MnSO4H2O, 0.1 g CuSO45H2O and
0.1 g Na2MoO42H2O.
Isolation of pectin-degrading microorganisms
Plant material (flax or hemp) was cut into pieces and
0.5 g of it was incubated in 50-mL hemp pectin
medium at 65C for 2 days. One hundred microlitres
of the obtained suspension were spread on plates and
pectin-degrading microorganisms were isolated follow-
ing incubation at 65C.
Cultivation of pectin-degrading organisms
Cultivations in shake flasks with baffles in 100-mL
minimal medium were carried out to prepare inocula for
Appl Microbiol Biotechnol (2009) 83:521527
fermentations. Fermentations of the isolates were carried
out in a stirred 2-L bioreactor and a 20-L bioreactor
(Bioengineering AG, Wald, Switzerland) in minimal medi-
um. The reactor was aerated with 0.1 vvm and the pH was
kept at 8 by the addition of 0.1 M NaOH.
Plate test for cellulase activity
Cellulase tests were done with a modified technique that uses
Congo red to characterise cellulolytic bacteria (Teather and
Wood 1982) as follows: 0.8% (w/v) agar and 0.5% (w/v)
carboxymethylcellulose were added to 100-mL demineral-
ised water, boiled and poured into Petri dishes. Wells of
8-mm diameter were made into the solidified plates and
80 L of culture supernatant or commercial enzyme was
filled into them. After incubation at 65C, the plates were
covered with 15-mL 0.1% Congo Red solution for 15 min at
4C and washed with 1-M NaCl solution for 15 min.
Cellulose-degrading activity was detected by yellow halos in
the stained cellulose plates. Commercially available cellulase
was used as positive control.
Plate test for pectinase activity
The tests were done with a modified assay for the quantifi-
cation of polysaccharide-degrading enzymes (Brhlmann et
al. 1994) as follows: 0.5% agarose (w/v) and 1% citrus pectin
(w/v) were dissolved in 100-mL mineral medium for
denitrifying bacteria, modified from Atlas (2004). The pH
was adjusted to 8 with NaOH and the solution was boiled
and poured into Petri dishes. Eight-millimetre-diameter wells
were made into the solidified plates and filled with culture
supernatant or enzyme. After incubation at 65C, the plates
were covered with 15-mL 0.05% Ruthenium Red solution
for 30 min at 4C and then washed with distilled water.
Pectin-degrading activity was detected by clear halos in the
stained plates. Water was used as negative control.
Enzymatic hemp fibre treatment
The 12.5-g hemp fibres were washed thoroughly, soaked in
350-mL tap water for 0.5 h and washed again with warm
water. After the washing, the fibres were put into 350-mL
culture broth or enzyme preparation in a 500-mL beaker and
incubated at 60C in a rotary shaker bath at 160 rpm for
18.5 h. After incubation, the culture broth was removed and
fibres were washed thoroughly with warm water and dried
overnight at 37C.
Determination of the uronic acid content of hemp fibres
Uronic acid was detected by the method of Blumenkrantz
and Asboe-Hansen (1973). Ten grammes of hemp fibres
523
were autoclaved in 200 mL of 1% (v/v) sulfuric acid. The
extract was filtered, and water was added to 250 mL. This
solution was diluted 1:20 with demineralised water. Two
hundred microlitres of this solution were boiled for 10 min
with 1.2 mL 12.5 mM sodium tetraborate in 97% sulphuric
acid in a glass tube. After boiling, the mixture was cooled
on ice and 20 L of m-hydroxydiphenyl-sodium hydroxide
(0.15% (w/v) MHDP in 0.5% (w/v) NaOH) solution was
added. The tubes were incubated for 10 min on ice.
Absorption was measured at 520 nm. Galacturonic acid
dilutions were used as standards to calculate the content of
uronic acid in the fibres.
Pectin lyase assay
Pectin lyase activity was determined in the crude enzyme
extract, which was obtained by centrifugation of the culture
broth. The activity was determined spectrophotometrically
by measuring the increase of absorbance at 235 nm due to
-elimination and formation of double bonds in solutions
with pectic substances as substrate according to a modified
procedure of Collmer et al. (1988).
From the crude enzyme extract, 150 L was mixed with
1,275 L of buffer (0.1-N aqueous glycine solution with
0.68 mM CaCl2 adjusted to pH 10 with NaOH). In a cuvette
at 60C, 950 L of this mixture was mixed with 50-L
substrate solution (0.5 g pectin in 50-mL glycine buffer,
heated to 60C and centrifuged to remove undissolved
material). Absorption changes were measured at 235 nm
for 5 min. One unit of enzyme activity was defined as the
amount of enzyme necessary to form 1 nmol/min of
unsaturated oligogalacturonides. The molar extinction coef-
ficient of 5,500 per molar per centimetre was used (van
Alebeek et al. 2002).
Concentration of enzyme solutions
Supernatants from cultivation broths containing pectin
lyase activity were concentrated using an ultrafiltration
hollow-fibre module with a cutoff of 10 kDa from
Amersham Biosciences, Freiburg, Germany.
Measurement of fibre fineness with optical-based fibre
diameter analyser
Dry fibres were stored in an air-conditioned room at 20C
and 65% humidity for at least 24 h. Small samples of the
fibres were placed on glass plates and the width of fibres or
fibre bundles were analysed optically at different areas of
the sample. If the fibre width was between 4 and 150 m,
the fibre was counted and arranged into width classes. Per
sample, 22,000 single-width class measurements were
carried out and the analysis was done by the software of the
524
optical-based fibre diameter analyser (OFDA) device.
These measurements were carried out at the FIBRE
Institute, Bremen.
Accession number for G. thermoglucosidasius PB94A
(DSM 21625) at NCBI is FJ491390 and for G. thermode-
nitrificans PB1511 (DSM 21923) is FJ491391.
Results
Isolation of the new hemp pectin-degrading bacteria
For the screening of hemp pectin-degrading microorganisms,
hemp medium was used and different samples of flax and
hemp served as microorganism sources. Screening was carried
out at 65C. Cultures were obtained using standard enrich-
ment culture techniques on medium with pectin as the sole
carbon source at pH 8 and 65C. Two isolates reached the
highest cell densities when grown in liquid media with hemp
pectin. The new isolates shown in Fig. 1 were identified as G.
thermodenitrificans PB1511 (DSM 21923) and G. thermo-
glucosidasius PB94A (DSM 21625) by the DSMZ
Braunschweig, Germany. Both strains are spore-forming
thermophilic bacilli of 0.70.9-m width and 2.54.0-m
length. They produce acid from D-glucose, D-xylose, D-
mannitol and D-fructose. DSM 21923 also degrades L-
arabinose and citrate. The two strains utilise starch and
casein and reduce nitrate while only DSM 21625 degrades
gelatin. They show no phenylalanine desaminase and
arginine dihydrolase activity.
Hemp fibre processing with the new strains
Hemp fibres from partially field-retted hemp were
treated with cultures of the new isolates, which were
grown on minimal medium with pectin as the sole
carbon source to different cell densities; negative
controls were done with water and minimal medium.
The treatment was done under non-sterile conditions in
a beaker on a rotary shaker for 18.5 h in culture broth.
Fig. 1 Microscopic pictures of
G. thermodenitrificans PB1511
(a) and G. thermoglucosidasius
PB94A (b)
Appl Microbiol Biotechnol (2009) 83:521527
After the treatment of the fibres, the residual pectin
content was determined by the analysis of the uronic
acid content of the processed fibres, which can be
linked to the extent that the pectin was degraded from
the fibres and therefore to their refinement. The raw
fibre uronic acid content was 3.2%, and the treatment
with water apparently increased the uronic acid content
by 6% probably due to the liberation of the pectin
during the incubation making it easier to detect. The
treatment with minimal medium removed 14% of the
uronic acid. A possible explanation for this reduction
could be the degradation by pectinolytic microorganisms
already present in the fibres, which was in fact observed
under the microscope.
G. thermodenitrificans PB1511 reduced the pectin
content of the fibres approximately 30% and G. thermo-
glucosidasius PB94A 45%. Therefore, the latter strain was
chosen for further investigations.
Detection of cellulase and pectinase activity in G.
thermoglucosidasius PB94A and Bioprep 3000 L
Since the new isolate, G. thermoglucosidasius PB94A,
should be used for bast fibre treatment, it was important
that it used pectin as a carbon source but showed no
cellulolytic activity. Otherwise, the cellulose fibres would
be damaged and weakened. Plate tests for cellulose-
degrading activity were carried out with carboxymethylcel-
lulose as substrate with cellulase from Aspergillus niger as
positive control. Figure 2a shows that the new strain
produced no cellulase. For comparison, plate tests were
also performed with the commercial enzyme Bioprep
3000 L to check for cellulolytic side activity. Figure 2b
shows that this commercial pectinase also degrades the
carboxymethylcellulose and thus has cellulase activity,
corresponding to around 10 U/mL.
Plate tests to detect pectinase activity of PB94A
fermentation broth and of Bioprep 3000 L were made.
Supernatant of PB94A fermentation broth showed degra-
dation of the pectin while incubation with sterile water
Appl Microbiol Biotechnol (2009) 83:521527 525

Fig. 2 Plate tests for cellulase

activity of a G. thermoglucosi-
dasius PB94A (well 1) in com-
parison to a commercial
cellulase at different concentra-
tions (wells 2, 3 and 4 corre-
spond to 0.8, 0.4 and 0.08 U,
respectively) and b Bioprep
3000 L containing 120 U
pectinase in the outer wells of
the plate; water was used at the
centre well of both plates as a
control

showed no degradation. In the case of Bioprep 3000 L, the
pectin was degraded almost on the entire plate.
Detection of pectinolytic enzymes from G.
thermoglucosidasius PB94A during growth on pectin
the fibre diameter of the raw material and of the processed
samples are shown in Fig. 4. Processing with higher
concentrated pectinolytic lyase solutions led to finer fibres
and the fineness of hemp fibre material could be improved
drastically by the enzymatic treatment.

Batch fermentations of G. thermoglucosidasius PB94A Substrate specificity of the pectin lyases

on minimal medium were carried out to determine the
growth characteristic of the isolate and to find out what Commercial enzyme formulations are available for the
types of pectinolytic enzymes were produced by this processing of natural fibres containing pectinases from
isolate being responsible for the pectin degradation during different sources (Dreyer et al. 2002; Sharma et al. 2005;
fibre processing. The isolate showed best growth at a Foulk et al. 2001; Akin et al. 2002; Zhang et al. 2005). One
temperature of 60C and pH 8. Assays of the culture of these formulations, Bioprep 3000 L (Novozymes A/S,
supernatant during fermentation with different pectic Bagsvrd, Denmark), has been used in hemp fibre
substrates as the sole carbon source showed that pectin processing before (Dreyer et al. 2002). It is an alkaline
lyases could be detected during the growth of the culture stable pectin lyase produced by Bacillus sp. and is used for
but no pectin hydrolases were detected on any substrate.
G. thermoglucosidasius PB94A grew well on glucose but
no pectinolytic enzymes could be detected in these
fermentations. Highest specific activity of pectinolytic
lyases was found in cultures grown on polygalacturonic
acid as the sole carbon source. Figure 3 shows the result of
a fed-batch fermentation. It can be clearly seen that the
production of pectinolytic lyase was growth-associated.
In a chemostat cultivation, a space time yield of 25.4 U/mL
per hour was reached using a dilution rate of 0.18 per hour
using dialysed citrus pectin as substrate.

Hemp fibre processing with different enzyme

concentrations

A batch cultivation of G. thermoglucosidasius PB94A was

carried out in a 20-L bioreactor on polygalacturonic acid as
substrate. The supernatant of this fermentation was con-
centrated to different concentrations of pectinolytic lyase.
These solutions were used to process hemp fibres.
Treatment was done for 1 h to avoid over-retting or
Fig. 3 Cell number (squares) and lyase activity (triangles) during
extraneous microbial degradation of the fibres. The fibre fed-batch fermentation of G. thermoglucosidasius PB94A on poly-
diameters were determined by OFDA. The mean values for galacturonic acid as substrate
526
Fig. 4 Fibre width of raw hemp and of hemp treated with PB94A
enzymes at different concentrations
biological retting and scouring of cotton fibres. The
substrate specificity of the purified pectin lyases produced
by G. thermoglucosidasius PB94A was compared to the
Bioprep 3000 L. For the substrate specificity test, pectins
from different origins were used as substrates in the pectin
lyase assay. The results are shown in Fig. 5.
G. thermoglucosidasius enzymes and Bioprep 3000 L
were most active towards apple pectin as a substrate and
therefore all other values were normalised with this
reference.
The activity with citrus pectin and sugar beet pectin
is similar for PB94A enzymes and Bioprep 3000 L.
With polygalacturonic acid, Bioprep 3000 L showed
about three times the activity of PB94A enzymes. With
hemp pectin, Bioprep 3000 L showed no activity at all,
while PB94A enzymes showed similar activity as with
polygalacturonic acid.
Discussion
So far, several Bacillus strains have been reported to
produce pectinolytic enzymes (Cao et al. 1992; Kobayashi
et al. 1999; Ogawa et al. 2000; Soares et al. 1999; Takao et
al. 2000; Yavuz et al. 2004; Zhai et al. 2003). To our
knowledge, no pectinolytic enzymes have been reported for
the genus Geobacillus, which was recently reclassified
from bacillus (Nazina et al. 2001).
The 16S rDNA sequence and fatty acid analysis
determined at DSMZ showed that the strain belongs to
the genus Geobacillus. The 16S rDNA was 100% identical
to G. thermoglucosidasius. Therefore, the strain was named
G. thermoglucosidasius strain PB94A
The ability of PB94A to grow at 65C and pH 8 is an
advantage for use in fibre retting because the risk of
contamination by cellulolytic microorganisms is almost
eliminated. The strain is aerobic, and this factor allows the
Appl Microbiol Biotechnol (2009) 83:521527
development of a process without the inconveniences of
malodour from anaerobic ones.
Even though pectin is a very complex substance, which
varies widely in its composition (Vignon and Garca-Jaldon
1996), the enzymes from G. thermoglucosidasius PB94A
are sufficient to reduce the pectin content in hemp fibres,
despite the fact that, in nature, several microorganisms are
responsible for plant retting (Sharma et al. 1992).
The removal of pectin from the fibres can be done with a
few enzymes because it is not necessary to degrade the
pectin to monomers but just to cleave the pectin at some
positions as reported by Zhang et al. (2005).
The results shown in Fig. 5 provide some information
about the types of lyases produced by G. thermoglucosi-
dasius PB94A. Since activity with polygalacturonic acid
could be determined, a pectate lyase must be present in the
fermentation broths. The activity with polygalacturonic acid
is much lower than with the esterified pectins, which
suggests that there is a pectin lyase present as well. Pectin
from hemp fibres contains rhamnogalacturonan (Cronier et
al. 2005), which could indicate the existence of rhamnoga-
lacturonan lyases in G. thermoglucosidasius. Rhamnoga-
lacturonases have been described to degrade the hairy
regions of pectin (Schols et al. 1990). However, we were
not able to detect such an activity due to the lack of suitable
substrates. Since the broths show the same activity towards
polygalacturonic acid and hemp pectin, either the enzyme
has a broad substrate specificity or the expression of pectate
lyase and rhamnogalacturonan lyase is similar.
In chemostat cultures, the space time yield of pectino-
lytic lyases was increased in comparison to fed-batch
fermentation; therefore, continuous cultivation of G. ther-
moglucosidasius PB94A should be examined further. It
would be advantageous to increase the yield of pectinolytic
lyases in cultivations of G. thermoglucosidasius PB94A
further since a clear effect of enzyme activity on fibre
Fig. 5 Substrate spectrum of the lyase from G. thermoglucosidasius
PB94A (grey bars) in comparison to Bioprep 3000 L (white bars)
with different pectin types and polygalacturonic acid as substrates
Appl Microbiol Biotechnol (2009) 83:521527
fineness was observed. When solutions with higher enzyme
activity were used for fibre processing, finer fibres were
obtained. The fibres processed with high enzyme activities
reached fibre diameters which come close to the diameters
for the single cellulose fibres existing in the plant,
previously reported to be around 25 m (McGovern 1983).
Further investigations have to be carried out to character-
ise the enzymes in detail and to optimise the fermentation
conditions. However, we think that the new strain G.
thermoglucosidasius PB94A and its enzymes show a
promising potential for the processing of bast fibres.
Acknowledgements This study was supported by the Bundesministe-
rium fr Bildung und Forschung with the project 0312640A and by the
Fachagentur Nachwachsende Rohstoffe with the project 22006303. We
thank Dr. rer. nat. Holger Fischer and Dr. Jrg Mssig from Faserinstitut
Bremen (FIBRE) for the analysis of the fibre fineness.
References
Akin DE, Slomczynski D, Rigsby LL, Eriksson KEL (2002) Retting
flax with endopolygalacturonase from Rhizopus oryzae. Text Res
J 72(1):2734
Atlas (2004) Handbook of microbiological media. CRC, Boca Raton
Blumenkrantz N, Asboe-Hansen G (1973) New method for quantita-
tive determination of uronic acids. Anal Biochem 54(2):484489
Brhlmann F, Kim KS, Zimmerman W, Fiechter A (1994) Pectinolytic
enzymes from actinomycetes for the degumming of ramie bast
fibers. Appl Environ Microb 60(6):21072112
Cao J, Zheng L, Chen S (1992) Screening of pectinase producer from
alkalophilic bacteria and study on its potential application in
degumming of ramie. Enzyme Microb Technol 14:10131016
Collmer A, Ried JL, Mount MS (1988) Assay methods for pectic
enzymes. In: Wood WA, Kellogg ST (eds) Methods in
enzymology, vol 161. Academic, New York, pp 329335
Cronier D, Monties B, Chabbert B (2005) Structure and chemical
composition of bast fibers isolated from developing hemp stem. J
Agric Food Chem 53:82798289
Dreyer J, Mssig J, Koschke N, Ibenthal W-D, Harig H (2002) Comparison
of enzymatically separated hemp and nettle fibre to chemically separated
and steam exploded hemp fibre. J Industrial Hemp 7(1):4359
Foulk JA, Akin DE, Dodd RB (2001) Processing techniques for
improving enzyme-retting of flax. Ind Crop Prod 13:239248
Kashyap DR, Vohra PK, Chopra S, Tewari R (2001) Applications of
pectinases in the commercial sector: a review. Bioresource
Technol 77:215227
Kessler RW, Becker U, Kohler R, Goth B (1998) Steam explosion of
flaxa superior technique for upgrading fibre value. Biomass
Bioenerg 14(3):237249
Kluskens LD, van Alebeek GJWM, Voragen AGJ, de Vos WM, van
der Oost J (2003) Molecular and biochemical characterization of
the thermoactive family 1 pectate lyase from the hyperthermo-
philic bacterium Thermotoga maritima. Biochem J 370:651659
Kobayashi T, Hatada Y, Higaki N, Lusterio DD, Ozawa T, Koike K,
Kawai S, Ito S (1999) Enzymatic properties and deduced amino
acid sequence of a high-alkaline pectate lyase from an alka-
liphilic Bacillus isolate. Biochim Biophys Acta 1427:145154
McGovern JN (1983) Fibres (vegetable). In: Grayson M (ed)
Encyclopedia of composite materials and components. Wiley-
Interscience, New York, pp 497512
527
Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA,
Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS,
Ivanov MV (2001) Taxonomic study of aerobic thermophilic bacilli:
descriptions of Geobacillus subterraneus gen. nov., sp. nov. and
Geobacillus uzenensis sp. nov. from petroleum reservoirs and
transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus,
Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermo-
glucosidasius und Bacillus thermodenitrificans to Geobacillus as the
new combinations G. stearothermophilus, G. thermocatenulatus, G.
thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G.
thermodenitrificans. Int J Syst Evol Micr 51:433446
Ogawa A, Sawada K, Saito K, Hakamada Y, Sumitomo N, Hatada Y,
Kobayashi T, Ito S (2000) A new high-alkaline and high-
molecular-weight pectate lyase from a Bacillus isolate: enzymatic
properties and cloning of the gene for the enzyme. Biosci
Biotechnol Biochem 64(6):11331141
Sanio C (1995) Zur Mikrobiologie der Flachsrste: bakteriologische,
biochemische und technologische Untersuchungen. Dissertation
Westflische Wilhelms-Universitt Mnster, Germany
Schols HA, Voragen AGJ (2003) Pectic polysaccharides. In: Whitaker
JR, Voragen AGJ, Wong DWS (eds) Handbook of food
enzymology. Marcel Dekker, New York, pp 829843
Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, Kormelink FJM,
Voragen AGJ (1990) Rhamnogalacturonase: a novel enzyme that
degrades the hairy regions of pectins. Carbohyd Res 206:105115
Sharma HSS, Lefevre J, Boucaud J (1992) Role of microbial enzymes
during retting and their effect on fibre characteristics. In: Sharma
HSS, van Sumere CF (eds) The biology and processing of flax.
M Publications, Belfast, pp 199212
Sharma HSS, Whiteside L, Kernaghan K (2005) Enzymatic treatment
of flax fibre at the roving stage for production of wet-spun yarn.
Enzyme Microb Technol 37:386394
Soares MMCN, da Silva R, Gomes E (1999) Screening of bacterial
strains for pectinolytic activity: characterization of the polyga-
lacturonase produced by Bacillus sp. Rev Microb 30:299303
Takao M, Nakaniwa T, Yoshikawa K, Terashita T, Sakai T (2000)
Purification and characterization of thermostable pectate lyase
with protopectinase activity from thermophilic Bacillus sp. TS
47. Biosci Biotechnol Biochem 64(11):23602367
Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide
interactions in enumeration and characterization of cellulolytic
bacteria from the bovine rumen. Appl Environ Microb 43(4):777780
Van Alebeek G-JWM, Christensen TMIE, Schols HA, Mikkelsen JD,
Voragen AGJ (2002) Mode of action of pectin lyase A of
Aspergillus niger on differently C6-substituted oligogalacturo-
nides. J Biol Chem 227(29):2592925936
Van Sumere CF (1992) Retting of flax with special reference to enzymatic
retting. In: Sharma HSS, van Sumere CF (eds) The biology and
processing of flax. M Publications, Belfast, pp 157198
Vignon MR, Garcia-Jaldon C (1996) Structural features of the pectic
polysaccharides isolated from retted hemp bast fibres. Carbohyd
Res 296:249260
Von Drach V, Klo KD, Hensel K-H (1999) Ultraschallbehandlung von
PflanzenfasernHerstellung und Einsatz. In: 2nd International Wood
and Natural Fibre Composites Symposium, Kassel, vol. 23, pp 128
Yavuz E, Gunes H, Harsa S, Yenidunya AF (2004) Identification of
extracellular enzyme producing thermophilic bacilli from Bal-
cova (Agamemnon) geothermal site by ITS rDNA RFLP. J Appl
Microbiol 97:810817
Zhai C, Cao J, Wang Y (2003) Cloning and expression of a pectate
lyase gene from Bacillus alcalophilus NTT33. Enzyme Microb
Technol 33:173178
Zhang J, Henriksson H, Szabo IJ, Henriksson G, Johansson G (2005)
The active component in the flax-retting system of the zygomy-
cete Rhizopus oryzae sb is a family 28 polygalacturonase. J Ind
Microbiol Biotechnol 32:431438