Journal ofApplied Bacteriology 1978, 45,219-230

The Maceration of Linen Flax under Anaerobic

Conditions

A. CHESSON
Applied Biochemistry Division, Department of Scientific and Industrial Research,
Private Bag, Palmerston North, N e w Zealand

Received29 November 1 9 7 7 and accepted 14 December 1 9 7 7

Ret tanks of 350 1 capacity, loaded with 14 kg of flax straw and maintained at 32"C, were
sampled at regular intervals for 90 h. Total numbers of bacteria and viable anaerobes increased
rapidly during the initial 16 h, reached a maximum at 22 h and then remained constant in the ret
liquor. They continued to increase at a reduced rate within the flax straw. Dissolved oxygen
content and pH of the ret liquor fell rapidly over the initial period while volatile fatty acids,
ammonia and methanol accumulated. Xylanolytic and cellulolytic bacteria were detected in
increasing numbers after 40 h. Pectinesterase (PE.EC. 3.1.1.1 1) was restricted largely to the flax
straw where activity developed in parallel with microbial growth, reached a maximum at 22 h
and rapidly declined thereafter. Polygalacturonase (PG.EC. 3.2.1.15/40) and pectate lyase
(PAL.EC.4.2.2.1/2) activities developed after 16 h when soluble carbohydrates had been
depleted. Higher levels of both activities were associated with the flax straw than with the ret
liquor. P G predominated over PAL activity throughout. Other glycan hydrolyase activities were
not detected. Fractionation and analysis of straw samples showed that 83% of pectic
polyuronide present at the start of the ret was degraded by 70 h, whereas 16% of the hemi-
cellulose and none of the cellulose was lost over the same period.

IN THE RETTING of linen flax (Linum usitatissimum L.), bast fibre bundles are released
from the surrounding cortex by the action of pectic enzymes elaborated by a microbial
flora which develops during the process. Commercial retting is achieved by either (a)
dew retting in which straw, thinly spread on the ground, is exposed to the action of fungi
and aerobic bacteria for three to eight weeks, or (b) tank retting in which straw is sub-
merged for three to five days in warm water during which time a predominately
anaerobic population of bacteria develops.
Although the general principles underlying the retting of linen flax have been re-
cognized for many years (Dujardin 1948) there is a need for a reappraisal of the process
in the light of information gained from studies of the maceration of other plant
materials, notably in phytopathological conditions. Clostridium felsineum in pure
culture is the only organism isolated from a ret tank which has been examined in detail
for pectic enzyme production (Kapitonova et al. 1974; Avrova & Goshko (1975). Little
or no attention has been paid to the identification and levels of activity of pectic
enzymes produced by the mixed populations typical of industrial rets, nor to the
presence of other glycan hydrolyase activities and their possible contribution to
maceration.
Research involving tank retting has tended to concentrate on the identification of
bacteria responsible for retting (Rosemburg 1965), the application of pure cultures of
bacteria or fungi to accelerate the retting process (de Franca et al. 1969; Avrova 1975)
002 1-8847/78/020219+ 12 %Ol.OO/O [2191 0 1978 The Society for Applied Bacteriology
220 A. CHESSON

or attempts to identify suitable chemical or biological parameters which could be used

to determine the end-point of a ret (Rosemburg & de Franca 1967). These investiga-
tions have concentrated on the ret liquor surrounding the straw and ignored events
occurring within the straw. Yet when the dried straw is submerged there is a passive
uptake of organisms through stomata and damaged portions of the stem. It is these
organisms, in close proximity to their substrate, which are largely responsible for
maceration (Hadfield 1953).
The present work was undertaken to identify the pectic enzymes and other poly-
saccharide degrading activities present in industrial ret tanks, to determine their levels of
activity in both the ret liquor and the straw and to examine their effect on the poly-
saccharide composition of the flax straw.

Materials and Methods

Experimental retting conditions
Experimental retting facilities were provided by the Linen Flax Corporation of New
Zealand at Geraldine. These consisted of eight circular concrete tanks (61 cm i.d. x 1 14
cm) each capable of holding ca. 350 1. The tanks were arranged in two groups of four,
each group surrounded by an independently controlled water bath set for the duration
of the experiments at 32-2 & 1 O C .
A bulk sample of flax straw (L. usitatissimurn var. Reina), harvested from a single
paddock in 1976 and known to be of average fibre content, was retained and used
throughout our trials. Bundles (14.0 kg) of straw arranged as five sheaves (‘beets’) were
loaded into single tanks in an upright position. Small straw samples (12 cm in length) of
known weight (10 g) which could be removed from the tanks in the course of a ret with-
out unduly disturbing the ret liquor, were prepared beforehand from the entire length of
the flax straw and distributed amongst the sheaves in each tank. The tanks were filled
with cold water (leach water). After 2 h the tanks were drained and refilled with warm
water at 32 OC (ret water) and the ret commenced. Although on occasions tanks were
sampled for 90 h, retting was judged complete and the tanks emptied after approx. 70 h
(the technical end of the ret).
Sampling
Samples of ret liquor and two straw samples were taken from each tank at regular
intervals after the addition of the ret water. Ret liquor (approx. 1 1) was siphoned from
the centre of the tank at a point 70 cm below the surface of the liquor and collected
under CO,, the first 250 ml being discarded.
One straw sample (1 0 g) was homogenized with 200 ml of ret liquor for two min in a
Waring Blender the headspace of which was filled with CO,. The ratio of straw weight
to volume of ret liquor was equivalent to that found in the ret tanks. Supernatant from
the straw homogenate and the sample of ret liquor were used as follows: (a) 100 ml re-
tained for enzyme and soluble carbohydrate assay; (b) 50 ml acidified with 0.1 ml 5 ~ -
H,SO, for nitrogen and volatile fatty acid determinations; (c) 5 ml added to 5 ml 10%
(v/v) formalin for microscopic counts of bacteria, (d) 0.5 ml used as inoculum in a dilu-
tion series for viable anaerobic counts of bacteria. Samples (a) and (b) and the second
straw sample were immediately frozen in solid CO, and maintained in a frozen state
until taken to the laboratory for analysis.
 MACERATION OF LINEN FLAX 22 1

At least three tanks were sampled in this manner, often on separate occasions, and
the results expressed as the mean value obtained.
Media and culture conditions
Media were prepared and dispensed (4.5 ml) in roll tubes sealed with butyl rubber
stoppers following the methods of Hungate (1969). The reducing agent, cysteine
hydrochloride (0.3 g/l), and the redox indicator, Resazurin (0.002 g/l), were added to
all media and mineral salts solutions.
Counts of viable anaerobes were made by preparing serial dilutions of ret liquor or
straw homogenates in a mineral salts solution containing (g/l): CaCI,, 0.01; MgSO,.
7 H 2 0 ,0.01; KH,PO,, 0.05; K2HP0,, 0.05; NaHCO,, 0.5 and NaC1, 0.1. Disposable
syringes (1 ml) flushed with CO, were used to transfer 0-5 ml of appropriate dilutions to
roll tubes containing one or other of the following media which had been melted and
held at 5OoC in a water bath: (a) half-strength reinforced clostridial agar (RCA) or (b)
the mineral salts solution noted above supplemented with (g/l) yeast-extract, 1-0;
NH,Cl, 2.5; agar, 13.5. One of the following polysaccharides was included as sub-
strate: pectin, 0.5 (Brown-ribbon, Obipektin AG, Bischofzell, Switzerland): finely
ground xylan, 2.0 (Sigma Chemical Co.); or cellulose, 3.0 [prepared after Hungate
(1 950)l.
Dilution cultures were prepared in triplicate and counts, determined after incubation
at 35 OC, expressed as the mean number of colonies/ml. In some experiments Tween 80
(final concentration, 5 ml/l) was added to the media and to ret liquor before the straw
homogenates were prepared. Total cell counts were made using a counting chamber
(0.02 mm depth) and phase-contrast microscopy.

Enzyme assays
Polygalacturonase (PG) and pectate lyase (PAL) activities were determined by their
ability to release reducing groups from apple pectic acid (Obipektin AG) as detected by
the dinitrosalicylic acid (DNS) method of Miller (1 959). Centrifuged ret liquor or straw
homogenate (0.5 ml) was added to 0.5 ml of 1% (w/v) pectic acid in either McIlvaine’s
(1921) citrate-phosphate buffer pH 5.0 (PG) or 0.1 M-Tris-HC1 buffer pH 8.5 con-
taining 0.002 M-Ca2+(PAL) and incubated at 35 “C for 60 min. After addition of DNS
the developed colour was read at 575 nm against a zero time blank. Enzyme activity
was determined by reference to standard galacturonic acid solutions and the amount of
enzyme releasing 1 pmol reducing group/min under these conditions was expressed as
one unit of activity.
PAL activity was confirmed using the U.V.assay described by Albersheim & Killias
(1962) and the periodate-thiobarbituric acid assay of Rombouts (1972).
Pectinesterase (PE) was assayed by incubating ret liquor or straw homogenate (0.25
ml) with of 0.5% (w/v) brown-ribbon pectin (0.25 ml) in McIlvaine’s citrate-phosphate
buffer (pH 7.0). The reaction mixtures were incubated at 35 OC for 60 min and the
amount of methanol released determined by the method of Wood & Siddiqui (197 1).
The amount of enzyme releasing 1 pmol methanol/min under these conditions was
expressed as one unit of activity.
Glycan hydrolyase activities of ret liquor and straw homogenates were examined
with the following neutral sugar polymers: arabinan (Koch-Light Laboratories Ltd),
arabinogalactan (Aldrich Chemical Co. Ltd), carboxymethyl cellulose (BDH Ltd),
222 A. CHESSON

cellulose (Avicel and Whatman No. 1 filter paper), galactan (Koch-Light Laboratories
Ltd), soluble galactomannan and larchwood xylan (Sigma Chemical Co.). Substrates,
prepared as 0.5% (w/v) solutions in citrate-phosphate buffers (pH 3-10), were
incubated with an equal volume of ret liquor or straw homogenate at 35 "C. After 60
min, the reaction mixtures were assayed for the release of reducing groups by the
Nelson-Somogyi method (Somogyi 1952).

Analvsis of ret liquor

Total volatile fatty acids (VFA) were analyzed by steam distillation and titration of the
distillate with 0.04 M-NaOH. Individual VFA's present in the distillate were determined
by gas chromatography using a Varian Aerograph 660 with a column ( I .5 m x 3.0
mm id.) packed with 20% (v/v) neopentyl glycol adipate and 4% (v/v) H,PO, on 100-
120 mesh chromosorb W. Other relevant conditions were: column temperature, I10 "C
(isothermal): injector temperature, 150 "C;detector temperature, 160 "C; carrier gas
(N,) flow rate 7 ml/min.
Nitrogen was determined as ammonia before and after Kjeldahl digestion by an auto-
mated phenol-hypochlorite method (Brown 1973) using an AutoAnalyzer (Type I,
Technicon Instrument Corp., New York, U.S.A.). Dissolved oxygen in the ret tanks was
measured with a dissolved oxygen meter (54-ABP Yellow Springs Instrument Co.,
Ohio, U.S.A.) calibrated against f r e e water saturated with air. Soluble reducing sugars
were assayed by the method of Somogyi (1952) before and after hydrolysis with an
equal volume of M-H,SO, at 100 "C for 1 h, and soluble uronic acid by the method of
Blumenkrantz & Asboe-Hansen (1973). Methanol was determined by the method of
Wood & Siddiqui ( 1 97 1).

1 g sample refluxed for 5 min with 150 m l boiling 80% ( v h ) ethanol

I-+ Soluble sugars-discarded

Residue refluxed for 2 h w i t h 150 m l H,O at 100°C

Hot-water soluble pectic substances

Residue refluxed for 2 h with 150 m l 0 5% (w/v) ammonium oxalate at 100 C

Hot-oxalate soluble pectic substances

j.
Residue refluxed for 2 h with 1 5 0 m l 0 5 M-H,SO, at 100 C

1
. Hemicellulose sugars

Residue treated with 10 m l 72% (v/v) H,SO, at room temperature for 4 h. then diluted to 0 5
M-H,SO, and refluxed at 100 "C for a further 2 h

Cellulose sugars

'Lignin'

Fig. 1. Steps in the sequential fractionation of flax polysaccharides.

 MACERATION OF LINEN FLAX 223

Polysaccharide analysis ofjlax straw

Samples of frozen flaz straw were freeze-dried and then cut into approx. 1 mm lengths.
Weighed samples were sequentially extracted according to the scheme in Fig. 1.
The uronic acid content of the extracts was determined as galacturonic acid by the
method of Blumenkrantz & Asboe-Hansen (1973) and reducing sugars as glucose by
the Nelson-Somogyi method (Somogyi 1952). The proportion of polysaccharides
resistant to degradation by micro-organisms increased as retting progressed, and there-
fore values obtained for each fraction were corrected by reference to the ‘lignin’ content
of the straw at the start of the ret and the ‘lignin’ content of each sample.

Results
Growth of micro-organisms
Microscopic counts of bacteria and the numbers of viable anaerobic bacteria in the ret
liquor increased rapidly over the first 16 h. They reached a maximum at 22 h and then
remained constant or diminished slightly (Fig. 2). The pH of the ret liquor fell rapidly
over the first 22 h to a constant level of pH 4.60 after ca. 40 h. Dissolved oxygen values
also fell from 6.80 parts/106 0, at the start of the ret to 0.05 parts 0,/106 at 22 h and
to zero in subsequent samples.
During the initial period of growth, the bacterial numbers in straw homogenates
paralleled those in the ret liquor. After 22 h the numbers in straw homogenates con-
tinued to increase slowly whereas those in ret liquors remained constant. At 70 h, the
technical end of the ret, the difference in numbers was approximately four-fold.
Figure 2 gives the results obtained with RCA medium. Similar results were found
with pectin agar thus indicating that a majority of the anaerobic bacteria were capable
of utilizing pectin or its breakdown products. Bacteria which produced zones of clearing
in xylan or cellulose agar were rarely detected during the first 40 h of the ret but
increased thereafter and achieved populations of 2 x lo7 colonies/ml at 70 h in the
straw homogenates. Addition of Tween 80 to homogenates and media did not increase
the numbers of colonies developing in the media.

-
,o I/
40t Ib 20 o: 4b
Time (h)
20 do 710 810 ‘ 40

Fig. 2. Changes in pH, total number of organisms and total number of viable anaerobes in the
ret liquor (closed symbols) and straw homogenates (open symbols) during retting. 0 0 ,
viable anaerobe count/ml made in RCA; 0,total count/ml; A,pH o f ret liquors.
224 A. CHESSON

Technical end of ref

7
40

I I I U
10 20 30 40 50 60 70 00
Time (h)

Fig. 3 . ( a ) Changes in the concentrations of VFA’s in ret liquor (closed symbols) and straw
homogenates (open symbols) during retting. 0 0,total VFA; M, acetic acid; V,propionic
acid: A, butyric acid. (b) Changes in total soluble nitrogen (0 0 )and ammonia nitrogen
(D 0) in ret liquor (closed symbols) and straw homogenates (open symbols) during retting.

The leach water after 1 h contained 1 x lo6 viable anaerobes/ml, the counts being
obtained or1 RCA and pectin agar. Strains of bacteria present in the ret tanks were not
identified. Phase-contrast microscopic examinations revealed, however, that the
majority of organisms were rod-shaped and that endospores or spore-bearing rods
accounted for approximately 10% of the total population.
Flax straw, stained with 0.1% (w/v) acridine orange in 0.1 M-phosphate buffer pH
8.0 and mounted in immersion oil. were examined with a Reichert Zetopan microscope
using incident light at 415 nm (BG 12 exciter and 669 + 06515 barrier filters). The
surface of the secondarily thickened regions of the stem and fibre bundles themselves
were in the main free from micro-organisms. The epidermis and underlying tissue, which
were progressively degraded during retting and which could be readily stripped from the
remaining stem, were heavily populated. Squashed preparations of this portion of the
stem often appeared under the microscope as a monolayer of predominantly rod-
shaped organisms. Coccal forms, common in the ret liquor, were less often observed in
such preparations.
Although VFA (acetic, propionic and n-butyric acids) accumulated throughout the
ret, fermentation was most rapid between 16 and 27 h [Fig. 3(a)l. The VFAs listed
 MACERATION O F LINEN FLAX 225

above, remained in relatively constant molar proportions of 73 :6 :21 from 16 h onwards.

Valeric and caproic acids, which were noted by Whiting (1959) under similar retting
conditions, were not detected in our study.
Soluble nitrogen compounds were not detected in ret liquor at the beginning of the ret
but could be readily extracted from the straw [Fig. 3(b)l. They accumulated in the ret
liquor over the first 22 h but showed little increase thereafter. Ammonia, absent from
both ret liquor and straw homogenates at the start of the ret, increased rapidly in
concentration between 4 and 27 h, the period of active microbial growth and fermenta-
tion. The concentration of both VFA and ammonia in ret liquors and straw homo-
genates was very similar, presumably due to rapid diffusion between these two in the ret
tank.

Enzyme activity
PG, P A L and PE activities were all detected in the ret liquor and straw homogenates
[Fig. 4(a)]. Activity was greater in straw homogenates than in ret liquor. This was
particularly evident with PE activity.

I\ Technical end of ret

Or (6)
Technical end of ret

10 20 30 40 50
Time ( h )
60
- 70 60 90

.,
Fig. 4. ( a ) Changes in pectic enzyme activity in ret liquor (closed symbols) and straw homo-
genates (open symbols) during retting. # 0, PE activity; 0 0, PG activity; A A, PAL
activity. (b) Changes in the polyuronide composition of flax straw during retting. 0 ,hot-
water soluble fraction; hot-oxalate soluble fraction; A,0.5 M-H,SO, soluble fraction.
226 A. CHESSON

P G and PAL activities increased after 16 h corresponding to the cessation of micro-

bial growth in the ret liquor and the decrease in the rate of growth in the straw. As both
p-transeliminative and hydrolytic cleavage of a single linkage in polygalacturonic acid
results in the liberation of a reducing group, the use of a common substrate and assay
method for both activities indicated the relative importance of the two depolymerases
(measured at their optimum pH). PG activity was found at considerably higher levels
than PAL over the entire ret period.
The development of P E activity paralleled bacterial growth, reaching a maximum at
22 h; there was a rapid fall in PE activity thereafter. This fall did not appear to be due to
some artefact introduced by the assay method as standard methanol solutions pre-
pared in ret liquors assayed at the expected values.
With pectic acid as substrate, PG had an optimum p H of 5.0, which corresponded
closely with the p H of the ret liquor for much of the ret. PG activity was absent at pH
8 - 5 . PAL activity was optimum at p H 8.5 and absent at values below pH 6.0. In
common with the majority of bacterial endo-PALS, activity was dependent on calcium
ions and suppressed in the presence of 0.005 M-ethylenediamine tetraacetic acid.
Substitution of Brown-ribbon pectin (76% esterified) as substrate resulted in reduced
levels of activity in homogenates prepared from straw taken 40 h from the beginning of
the ret and in ret liquors at both p H 5.0 and 8.5.
The pH optimum of PE activity appeared greater than p H 8.0 but proved difficult to
determine precisely due to the presence of methanol released by alkaline saponification.
High activity was obtained at p H 7.0, however, and this pH was adopted for all P E
assays.
Although xylanolytic and cellulolytic bacteria were detected in appreciable numbers
in the latter stages of the ret, D-xylanase, carboxymethylcellulase (Cx) or cellulase
activities could not be detected in ret liquors freed from micro-organism or straw homo-
genates. Likewise no activity was detected against the other neutral sugar polymers
used in this study, although many of them are known to be components of pectic sub-
stances.
Polysaccharide degradation
Sample sheaves of straw were removed from the ret liquor at regular intervals and
extracted by the scheme given in Fig. 1. The entire lengths of individual stems were used
TABLE1
Changes in the gross carbohydrate composition o f j a x straw during retting
Carbohydrate and lignin composition
% dry wt

Technical
Untreated Start of ret end of ret Over-ret
Fraction straw (0 h) (70 h) (90 h)

Pectic polyuronide 3.87 3.79 (3.67)t 0.65 (0.72) 0.55 (0.61)

Hemicellulose sugars* 11.90 10.82 (10.79) 8.99 (9.92) 8.96 (10.00)
Cellulose sugars* 40.55 40.40 (40.28) 39.00 (42.99) 39.02 (43.55)
‘Lignin’ 15.05 15.05 (15.00) 15.05 (16.60) 15.05 (16.80)

* Anhydroglucose equivalent.
f’ Values in parentheses are uncorrected for equal lignin content.
 MACERATION O F LINEN FLAX 221

TABLE2
Residual uronic acid content offibre and shive* from straw retted f o r 70 h

Anhydrouronide content
% dry wt
A
I 7
Fibre Shive*
Fraction (31.9% of stem wt) (61.1% of stem wt) Total

Hot-water soluble 0.09t 0.08 0.17

Hot-oxalate soluble 0.19 0.18 0.37
Total pectic polyuronide 0.28 0.26 0.54
0.5 M-H,SO, soluble 0.38 2.10 2.48

* Residual stem debris after extraction of the fibre.

f Values corrected for equal lignin content.

to prepare samples and the results obtained represent the average rate of degradation. It
was generally found that the butt and middle of the straw retted more rapidly than the
head.
The changes in uronic acid content in the hot-water, hot-oxalate and hemicellulose
fractions during the course of the ret are shown in Fig. 4(b).A portion of the hot-wGer
soluble fraction was lost by leaching before appreciable pectinolytic activity was
detected. Further loss of polyuronide from this fraction occurred in the presence of
pectic enzyme activity but a substantial proportion (25%) of that present initially re-
mained after 40 h and was resistant to further breakdown. Polyuronide present in the
hot-oxalate soluble fraction was resistant to leaching but was degraded readily in the
presence of pectic enzymes. Eighty percent of the total uronide in this fraction was
degraded during the 20 h following the development of pectin depolymerase activity. A
residual part (1 5%) of this fraction also appeared to be resistant to pectinolytic action.
The major portion (83%) of the pectic polyuronide (considered as the sum of the hot-
water and hot-oxalate fraction) was lost over the ret period (Table 1). After 70 h, the
residual pectic polyuronide was found to be distributed unevenly within the stem.
Slightly over half was associated with the fibre although fibre represented only 32% of
the total weight of the straw (Table 2).
In marked contrast to the loss of pectic substances, there was only a 16% reduction
in hemicellulose sugars and no reduction in cellulose sugars over the same period. No
additional degradation of hemicellulose or cellulose polymers was detected after the
technical end of the ret (70-90 h) despite the presence of increased numbers of xylano-
lytic and cellulolytic organisms.
Soluble sugars released from the straw were detected in ret liquors and straw homo-
genates throughout the ret. Soluble uronic acid was found in ret liquors at a constant
but low level of 0.05-0.1 mmol/l. Higher concentrations (0.5 mmol/l) were detected
initially in the straw homogenates but decreased during the period of rapid bacterial
growth and increased again to 1.0 mmol/l at 27 h when pectinolytic activity developed.
Subsequent samples showed a steady decline in concentration falling to the level
detected in the ret liquor alone at 70 h.
Changes in the concentration of reducing sugars paralleled those of uronic acid but
were present overall at higher concentrations (0.2-0.5 mmol/l for ret liquor).
Hydrolysis did not increase appreciably the concentration of reducing groups.
228 A. CHESSON

Oligouronides and pseudoaldobiuronic acids would have been resistant to hydrolysis

under the conditions employed. Methanol, absent initially from the ret liquor and straw
homogenates, accumulated rapidly between 4 and 22 h.

Discussion
These results suggest that the maceration of flax straw may be conveniently considered
in three stages: the development of the anaerobic flora (0-20 h), a period of rapid
pectinolysis or maceration (20-50 h) and a period of slow pectinolysis (50 h to the end
of the ret).
During the initial stage of each ret the anaerobic population developed at the expense
of soluble carbohydrate and organic nitrogen compounds leached from the straw,
resulting in the accumulation of VFA’s and ammonia in the ret liquor. P E production
was also a characteristic of this phase of the ret. Activity was associated largely with
the straw and was found only at much lower levels in the ret liquor, suggesting that
production was induced. Clostridium felsineum has been shown to produce P E only in
the presence of high methoxyl pectin (Voznyakovskaya et al. 1974). The restriction of
high levels of P E to the initial stage of the ret has yet to be explained. Although a
number of factors may have been responsible for the loss of activity after 22 h, it would
be of interest to examine the effect of free methanol on the biosynthesis of this enzyme
by bacteria.
The rapid fall in P E activity as depolymerase activity developed and the early libera-
tion of methanol also suggested that de-esterification largely preceded cleavage of the
polyuronide chain. This observation is in contrast to the coupled de-esterification and
cleavage of glycosidic bonds noted with the complex formed by PE and exo-PAL
derived from Cl. multifementans (Lee et al. 1970).
The second stage was marked by the cessation of microbial growth in the ret liquor, a
considerable reduction in the rate of growth of organisms associated with the straw and
by the development of pectin depolymerases with preferential activity against
demethylated polygalacturonic acid. PG and PAL activities developed when the
leached, soluble carbohydrates had been depleted, possibly due to the lifting of
catabolite repression as has been previously described for P A L production by strains of
Erwinia (Starr & Chatterjee 1970) and Aeromonas (Hsu & Vaughn 1969).
PG activity, with an optimum p H close to that of the ret liquor, appeared to pre-
dominate throughout the ret although PAL is generally considered to be more
characteristic of bacterial pectic enzymes (Rombouts 1972). Bateman (1976) has noted
that, when phytopathogens are capable of elaborating both hydrolase and lyase
activities, the type of activity predominating is often related to the gross p H of the
environment.
The development of the pectic enzymes was not accompanied by the production of
other glycan hydrolases, although such activities have been commonly demonstrated in
other plant macerations (see Calonge et al. 1969; Jones et al. 1972). Kaji et al. (1963)
have also described the production of L-arabinanase activity by a pectinolytic strain of
Cl. felsineum. Thus the retting of linen flax, at least under the conditions described in
this paper, provides a further example of the ability of pectic enzymes alone to effect
cell separation.
 MACERATION OF LINEN FLAX 229

The levels of pectic enzyme activities associated with the straw were considerably
higher than those in the ret liquor. Calculated on the basis of the dry weight of the
straw, P G was present in the order of 2000 units/g and PAL at 1000 units/g. As micro-
scopic examinations indicated that the heavily lignified regions of the stem remained un-
colonized whereas the primary walled tissue of the cortex was heavily colonized, these
values undoubtedly underestimate the levels found in the latter, highly localized environ-
ment colonized by organisms within the straw.
Although there was a considerable reduction in the level of polyuronide within the
straw following the development of depolymerase activity, 17% of that initially present
appeared to be resistant to enzyme attack. The limitation of further degradation was
probably related to the presence of lignin. Differences between the hot-water and
oxalate-soluble polyuronide fractions in susceptibility to degradation by PG and PAL,
acting alone and in concert with PE, have yet to be established.
The lo& of 18% of the uronide initially present in the hemicellulose fraction during
retting is probably an indication of the imperfect fractionation of the sample into pectic
and hemicellulose polymers. Hemicellulose uronide, largely glucuronic acid residues, is
commonly present as side chain components of highly branched acidic xylans and, in
the absence of hemicellulase activities, is unlikely to be solubilized.
Although retting was judged complete after 70 h, much of the length of each straw
was satisfactorily retted by 50 h, the end of the second stage. The duration of the third
phase represented a compromise between over-retting the main portion of the stem and
the time required to complete the retting of the head. During this period there was a
slow degradation of the remaining polyuronide in the straw, which if allowed to
progress too far led to the partial separation of fibre cells with the consequent
weakening of the final product.
A pronounced increase in the numbers of organisms capable of utilizing cellulose and
xylan occurred during this third stage, although cellulase and hemicellulase activities
were not detected in ret liquors freed from micro-organism or straw homogenates and
no evidence of the degradation of these polymers within the straw was obtained. It
would appear that these activities were not functionally significant in the rets examined.
Results obtained in the course of this investigation suggested that the straw formed
an environment within the ret tank which was able to support higher populations of
micro-organisms and which was subjected to higher levels of enzyme activity than
might be presumed from measurements made on the ret liquor alone. The pectic
enzymes produced by the mixed bacterial population were only partially characterized.
Further detailed descriptions require the purification of the individual activities present.
A detailed comparison of the microbial populations from the straw and ret liquor would
also be of considerable interest,
I am grateful to Mr B. McIsaac, Manager of the Linen Flax Corporation of New
Zealand for providing facilities for this work, Drs R. T. J. Clarke and T. Bauchop for
their comments and advice, Dr Clarke for suggesting the use of acridine orange and Ms
B. M. Anderson for skilled technical assistance.

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