FIELD OF THE INVENTION
The present invention relates to microbial retting of major cultivated species of jute plants
in genus Corchorus (including Corchorus capsularis and Corchorus olitorius). In particular,
the invention is based on selective bacterial consortium(s) that are naturally selected to
form multi-species biofilm community on water submerged jute stems and perform
enzymatic retting. The selected bacterial consortium is designed to incorporate higher
numbers of pectinases producers; comparatively lower numbers of cellulase and xylanase
producers and no laccase producers. The invention is further targeted at providing resultant
enzymes and /or mixtures thereof capable of performing efficient jute-bast maceration,
without damaging the lignocellulosic fibers. Advantageously, upon application, the selective
bacterial consortium efficiently colonizes jute (stem) surfaces and forms biofilms on jute
stem surfaces. The biofilm based consortium firmly attach to jute stems and thereby confers
natural resistance to the application against 'wash-out' or dilution when retting is performed
in large water bodies and/or subject to accidental flooding/rain. Further, the biofilm form of
the consortium is directed to produce higher levels of retting enzymes (than their planktonic
form) and deliver these at the jute (stem)-biofilm microenvironment. The target specific
enzyme provision and delivery allows efficient retting, improved fiber quality, and reduced
retting time-period. The bacterial components of the consortium are not reported
pathogens/opportunistic pathogens. Further, the invention is applicable for both the
conditions of retting , i.e. under 'contained' conditions (closed water bodies or tanks) and/
or simple rural retting techniques.
BACKGROUND ART
It is well-known that the most common method for production of plant bast fibers is
'Retting'. In particular such retting involves the degradation of the cementing material in
plant bast/stems thereby allowing extraction of the embedded fibers therein. Such retting is
known to be achieved following physical, chemical or biochemical/biological processes.
In case of physical retting the plant bast is passed through mechanical system to separate
the soluble cementing material. Chemical retting involves use of combination of acid and
alkali to solublize/dissolve the cementing material. In case of biochemical/biological retting
the same involve enzymatic digestion of the plant bast for degradation of its cementing
material.
2

Importantly, such biochemical or biological retting is achieved through two major routes:
(1) one or more microbes in the form of a deliberately formulated inoculum is/are used to
grow and proliferate in the retting environment and perform retting and
(2) purified or semi-purified form(s) of the microbial derived (retting) enzymes are used for
degradation of the cementing material. The use of purified/semi-purified microbial
derived enzymes is obviously cost extensive and has limitation of wide scale application,
especially in economically backward, rural south-east Asia responsible for jute
production.
As regards the provision of pre-formulated microbial inoculum, two types of microbial
inoculums are reported. The first entails species of fungus i.e., Sporotrichum and Aspergillus
either independently or in consortium; in the second bacterial consortium are used.
CN1038316 provides for a quick process of retting flax with warm water involving fungal
innoculum. A fungus quick-growth agent is added into the flax-retting pond which is stated
to reduce the time of retting to about 1/3.
Fungal pectinases (and related bast degrading enzymes) are reportedly most effective at
low pH. Most natural water bodies are at neutral pH. The low pH ideal for fungal enzymes is
normally attained as fungal population grows on the retting ecosystem. However fungal
growth rate is slower than bacteria and the bacterial pectinases work most effectively at
neutral pH. Besides, acidity in retting water bodies is not amenable to normal flora,
estuarine systems and human workers exposed to retting water.
Biochemical/enzymatic retting is another reported method for jute and related bast fibers
production and involves direct addition of purified retting enzymes (also called macerating
enzymes), predominantly microbial derived, on target stems.
WO 94/013868 is directed to a method of retting bast fibres, especially linen, using at least
one enzyme capable of degrading at least the pectines contained in the natural fibre. The
method is characterized in that an enzymatic solution containing an endo polygalacturonase
(EC 3.2.21.15) buffered by a buffer solution at a pH ranging from 3 to 6 is used to act on
3

the fibre. The present invention also concerns an enzymatic solution for retting bast fibres,
especially linen, characterized by comprising an endo polygalacturonase (EC 3.2.1.15).
WO 95/016808 discloses an industrial method for retting bast fibre plants such as flax by
means of the following enzymes: endopolygalacturonase (E.C.3.2.1.15), galactanases
(E.C.3.2.1.89, E.C.3.2.1.90), and xylanases (E.C.3.2.1.8; E.C.3.2.1.32).
US4891096 discloses a process for biochemical retting of phloem-fiber plants such as hemp,
flax, ramie, jute, kenaf, etc. permits separation of the fibers of the bundle for subsequent
utilization in the textile or paratextile industry. The process comprises treating the fibrous
plants with at least one SPS-ase enzyme and preferably a mixture of enzymes comprising
.beta.-glucanase, pectinase and SPS-ase.
US 2004/0191888 discloses a method for separating bast fibres by maintaining the
decorticated bast skin in a closed container (gas impermeable) with chemical/ enzymatic
retting agents for a sufficient period to enable at least a portion of the fibre bundles to
separate into individual fibres.
US 2005/0250181 is directed to variants of microbial cell- wall degrading enzymes, more
specifically, to variants of enzymes having a pectinase structure similar to that of Bacillus
licheniformis enzymes exhibiting pectate lyase activity as their major enzymatic activity in
the neutral and alkaline pH ranges ; to a method of producing such enzymes ; and to a
method of using such enzymes in the textile, detergent and cellulose fibre processing
industries.
US 7273745, relates to bacterial pectinases and identification of DNA sequence of such an
enzyme from Bacillus sp.. The identified DNA was then incorporated in plasmid (pSJ1678),
which was finally transformed into a strain of E. coli. The specific pectinases produced from
the transformed is stated to have use in textile, detergent and cellulose fibre processing
industry.
The above state of the art of fibre processing including jute retting involving either (i)
microbes in the form of a deliberately formulated inoculums and perform retting or (ii)
purified or semi-purified form(s) of the microbial derived (retting) enzymes are used for
degradation of the cementing material. The use of purified/semi-purified microbial derived
enzymes is obviously cost extensive especially in economically backward, rural south-east
Asia responsible for jute production. There is thus a continuing need in the art to make such
4

fibre processing such as jute retting more simple, industrially adaptable and cost-
effective/commercially viable by way of paving the way for a faster ,effective, consistent
and eco-friendly retting.
OBJECTS OF INVENTION
It is thus the basic object of the present invention to provide a microbial composition
formulated to specially colonize target (jute) stems by forming biofilms on stem surface;
produce an unique combination of retting enzymes in sessile mode and specifically deliver
these enzymes at the biofilm-jute microenvironment - resulting in faster retting, better fiber
quality and less threat to environment, ecosystem and human health - than conventional
retting.
Another object of the invention is to comply or augment organic jute fiber production
standards avoiding the use of harmful chemicals based on a natural sourced bacterial
consortium .
Another objects of the present invention is directed to advancement in the retting of bast
fibers such as jute which would be simple and cost effective and favour application in rural
areas involving very little financial, technical and infrastructure investment which would
thus benefit jute producers across the world.
A further object of the present invention is directed to advancement in retting of bast fibers
such as jute which would facilitate faster cycles of fiber production and enable wide scale
application and use of quality fibers in various fields of application of such fibers.
Another object of the present invention is directed to facilitate the retting of jute fibers by
substantial reduction in its production cycle maintaining uniformity in quality and
characteristic of the fiber produced to facilitate its industrial use and application such as
biocomposite production including but not limited to automobile industry, packaging
industry, bio-based structural composite material for housing and infrastructure industry
and wood-replacement furniture industry.
Yet another object of the present invention is directed to the advancement in the technology
relating to bast fiber/jute retting which can be advantageously utilized by small industry
5

such as those producing bast fiber - polymer extruded pellets for application in
biocomposite industry.
A further object of the present invention is directed to provision of microbial composition
capable of biofilm production useful in jute retting by way of potent extra cellular polymeric
substance forming ability which would allow the bacterial components to attach directly to
the target stems (as biofilm) and provide for benefit attributes including (a) confer
resistance to the application against dilution in large water bodies and/or accidental flooding
(b) confer tolerance to the application against extreme biochemical conditions of retting
environment usually manifested by low pH, low dissolved oxygen and presence of retting
by-products, (c) importantly colonizing effectively and efficiently on the target stems
ensuring better microbial functional for the retting process.
Yet another object of the present invention is directed to a bacterial biofilm based retting of
bast fibers such as jute wherein the bacterial enzymes, unlike fungal enzymes would exhibit
higher comparative activity under neutral pH, which is characteristic of natural water and
thereby work under conditions that is more safe for human and associated ecoystem.
Another object of the present invention is directed to a microbial formulation suitable for
jute retting which would favour production of biofilm involving more pectinase producers,
less cellulase and xylanase producers and no laccase producers such as to efficiently
degrade stem cementing material but not the embedded lignocellulosic fibers.
A further object of the present invention is directed to a microbial formulation generating
biofilm based retting of bast fibers which would be adapted for jute stems as well as ribbons
and can be adapted both for rural and/or industrial application to impart greater flexibility,
efficiency and at the same time economies the process of jute retting.
Yet, further objects of this invention are directed towards selective identification and
characterization of the specific bast fibre retting enzymes and identify the genes and the
DNA sequences encoding them which would enable providing for the desired selective
enzymes and/or combinations thereof involving more pectinase producers, less cellulase
and xylanase producers and no laccase producers for faster consistent and commercially
viable jute retting and the like.
6

Yet another object of the present invention is directed to towards selective identification and
characterization of the specific bast fibre retting enzymes and identify the genes and the
DNA sequences encoding them which can be incorporated in other microorganisms to
favour generation of desired retting enzymes/combinations thereof more specifically and in
greater amounts, facilitating larger and economic industrial use.
SUMMARY OF THE INVENTION
Thus according to the basic aspect of the present invention there is provided a microbial
formulation providing for biofilm production useful for retting of bast fibres such as jute
comprising:
biological bacterial constituents selected from one Acinetobacter sp., one Pantoea sp., one
Pseudomonas sp. and two Bacillus sp.in bacterial biofilm generating reagents and inoculum
media favouring Extracellular Polymeric Substance (EPS) forming ability.
In a preferred embodiment, biological constituents are selected from five bacterial species
i.e. a species of Acinetobacter sp., Pantoea agglomerans, , Bacillus pumilus, Bacillus subtilis
and Pseudomonas aeruginosa
In accordance to a preferred aspect, the microbial formulation providing for biofilm
production useful for retting of bast fibres is a bacterial consortium comprising a selective
combination of Acinetobacter sp., Pantoea agglomerans, and Bacillus pumilus. in bacterial
biofilm generating reagents and inoculum media favouring Extracellular Polymeric
Substance (EPS) forming ability.
Importantly, in the above microbial formulation the said bacterial constituents and inoculum
media are selectively provided such that the microbial biofilm produces high levels of
pectinases, low levels of cellulases and xylanses but no laccases.
In accordance with an aspect of the present invention the said bacterial bioflim generating
inoculum comprises a first reagent selected from Disodium hydrogen phosphate (Na2HP04),
Potassium dihydrogen phosphate (KH2P04), Sodium Chloride (NaCI) and Ammonium
Chloride (NH4CI); and a second reagent selected from Pectin and nutrient broth.
7

In accordance with another aspect of the invention , the said microbial formulation includes
selective biological bacteria constituent„selected from one Acinetobacter sp., one Pantoea
sp., one Pseudomonas sp. and two Bacillus sp., all of which adopts the microaerophilic
growth mode in the low oxygen tension to synergistically favour the retting micro-
environment.
More specifically, according to a preferred aspect the biological bacterial constituents
comprise three species, Acinetobacter sp., Pantoea agglomerans, and Bacillus pumilus
selected from the above mentioned five bacterial constituents, which also favour the
microaerophilic growth mode in the low oxygen tension to synergistically favour retting
micro-environment
Advantageously, in the microbial formulation of the invention the said inoculum media
comprises sterilized reagents with pH of 7.0 i.e. neutral pH, in the substrate media.
Preferably, the microbial consortium is obtained as a concentrate suitable for dilution with
water before use.
In accordance with another aspect of the invention the method for the manufacture of the
microbial formulation of the invention comprises:
dissolving pectin in water and adjusting the pH to 7.0;
introducing the substrate media;
adding sterilized portions of said first and second reagents;
adding bacterial constituents and subjecting to incubation to thereby provide the microbial
consortium in solution for retting of cellulose fibres.
Preferably, the said step of incubation is carried out at a temperature of 32 °C to 37 °C for a
period of about 3 days.
8

In accordance with another aspect of the invention there is provided a process for the
biochemical retting of bast fibres such as jute using the microbial formulation of the
invention which comprises:
spreading the jute stems on a flat surface after cutting and removal of the leaves;
providing the said microbial consortium after suitable dilution;
spraying the diluted microbial consortium on the jute stems;
air drying the stems thus treated;
assembling the stems in loose bundles;
submerging the microbial consortium treated stems in water and allowing the retting to take
place.
In the above process for the biochemical retting of jute for field (natural tank) retting the
said submerged stems are preferably covered to favour the retting process.
Advantageously, in the above process for the biochemical retting of jute the retting is
achieved much faster as compared to the conventional methods and is completed in about 7
to 10 days.
Preferably, in the above process of retting of the invention, the diluted microbial
formulation/consortium is spread on the jute using automated or manual devices.
The dilution of the concentrated microbial consortium is done such that 1 litre of
concentrated solution is used for 3000 to 6000 jute stems.
In accordance with another aspect of the invention there is provided a process for the
biochemical retting of jute for contained (solid structure tank) retting wherein
the air dried microbial consortium treated stems are bunched and the bunches are tied
down to rings ;
water is filled in the tank so as to keep the stems submerged ;
9

releasing selected amount of water from the tank and adding fresh water along with fresh
inoculum;
adjusting the pH of the system to 7.0 with sodium bicarbonate under vigorous agitation;
subjecting the system to further incubation until retting is complete.
In the above process after retting is complete the stems are released and mechanically
moved to another tank for washing to release the fibres which are dried.
In the above process after release of the fibres from the tank the corner vents of the
primary tank are opened to allow the sludge and spent liquor from the primary tank into the
effluent treatment tank whereby the primary tank is ready for subsequent use for retting.
The sludge in the effluent treatment tank is mixed with jute leaves and twigs stored for
compost formation followed with optional addition of diammonium phosphate (DAP) and
then subjecting to incubation with occasional stirring to obtain therefrom clarified water for
recycling in the process of retting.
Advantageously, the compost is subjected to drying in sun and packaged as organic
fertilizer.
In accordance with yet another aspect of the present invention there is provided a system
for carrying out contained retting of fibres and the like such as jute fibres comprising:
a retting tank;
a trickling filter tank; and
a water recycling tank ,
said retting tank providing retting of the fibres under action of microbial consortium
/enzymatic treatment ;
10

means for supplying the effluent from the retting tank to said trickling filter tank for
treatment of the effluent under trickling filter technique wherein the organic waste from the
bio process is converted to compost fertilizer;
said trickling filter tank operatively connected to said water recycling tank such that the
water from the trickling filter tank is recycled into the retting tank for subsequent cycles of
retting.
Advantageously, in the retting tank means preferably rings are provided to favour securely
tying the bundle of stems for retting with strings fro the purpose of the retting.
The trickling filter tank is preferably comprised of plurality of filter media preferably
comprising a top large sized stone chip filter media , an intermediate sand filter media and a
bottom smaller sized stone chip media.
The retting tank is operatively connected to the filter tank through an effluent transfer port
at the top while the filter tank is operatively connected to the water recycling tank by water
transfer ports near the base.
According to yet further aspect of the invention the same is directed to selective
identification and characterization of the specific bast fibre retting enzymes/combinations
thereof and identify the genes and the DNA sequences encoding them based on the
selective biological bacterial constituents of the invention selected from one Acinetobacter
sp., one Pantoea sp., one Pseudomonas sp. and two Bacillus sp preferably including
a species of Acinetobacter sp., Pantoea agglomerans, , Bacillus pumilus, Bacillus subtilis
and Pseudomonas aeruginosa which would enable providing for the desired selective
combination of enzymes involving more pectinase producers, less cellulase and xylanase
producers and no laccase producers for faster ,consisten and commercially viable jute
retting and the like.
Yet further aspect of the present invention is directed to towards selective identification and
characterization of the specific bast fibre retting enzymes/combinations thereof and
identify the genes and the DNA sequences encoding them based on the selective biological
bacterial constituents of the invention selected from one Acinetobacter sp., one Pantoea sp.,
one Pseudomonas sp. and two Bacillus sp preferably including a species of Acinetobacter
sp., Pantoea agglomerans, , Bacillus pumilus, Bacillus subtilis and Pseudomonas
11

aeruginosa which can be possibly incorporated in other microorganisms to favour
generation of desired retting enzymes more specifically and in greater amounts and eco-
friendly manner, facilitating larger and economic industrial use.
EXAMPLES:
The details of the invention, its objects and advantages are explained hereunder in greater
details in relation to non-limiting exemplary illustrations of the manner of obtaining the
microbial formulation of the invention and its application in retting of jute fibers.
EXAMPLE I :
The following exemplary illustration is directed to the development of the microbial
formulation adapted for biofilm generation in accordance with the present invention.
For such purpose, the biological constituents were selectively provided such as to favour the
required high levels of pectinase along with cellulase which is found beneficial for
degradation of the cementing material of the bast fibres without affecting the lignocellulosic
contents inside the bast fibres. The biological constituents thus identified for the purposes of
the microbial formulation are as detailed hereunder in Table I.
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TABLE I

Bacteria Isolated
from Biofilm
formation Retting Enzymes Growth
conditions Survival in
mixed
community
biofilms in
vitro and in
vivo, (days)

Pectinase Cellulase

Acinetobacter
sp. Indian
Jute
Retting
Ecosystem Yes ++ - Microaerophilic 15 +
Pantoea
agglomerans Indian
Jute
Retting
Ecosystem Yes ++++ - Microaerophilic
/ Anaerobic 15 +
Bacillus
pumilus Indian
Jute
Retting
Ecosystem Yes +++ ++ + Microaerophilic 15 +
The inoculum favouring the bacterial biofilm growth was obtained as discussed hereunder:
Reagent A) BSS Solution was provided comprising the following:
Disodium hydrogen phosphate (Na2HP04) - 0.6 g
Potassium dihydrogen phosphate (KH2P04) - 0.3g
Sodium Chloride (NaCI) - 0.05 g
Ammonium Chloride (NH4CI) - O.lg
Reagent B comprised:
Pectin - 0.8g
Nutrient Broth (Difco, USA standard) - l/4x
13

EXAMPLE II:
Under this example the method of field (natural tank) retting involving the microbial
formulation suitable for generation of biofilm in accordance with the invention was carried
out as detailed hereunder:-
i) Jute stems were cut, leaves removed and stems spread on a flat surface;
ii) the bacterial inoculum concentrate obtained under Example I above was diluted
1: 1000 in water before use;
iii) the diluted inoculum was spread on the jute stems using a common agricultural
shoulder mounted manual spring device (1 liter of concentrate solution used for
3000-6000 jute stems);
iv) the stems were next to dried for about 30 minutes;
v) the stems were then assembled in loose bunches of about 20;
vi) the stems were then submerged in water;
vii) subsequently the stems (Jak) were covered with a plastic sheet;
viii) optionally , after about 3 days about 1 kg of Sodium Bicarbonate (common soda)
was dissolved in water was dispersed in the Jak and shaken well. Retting was
finally over in about 7-10 days.
EXAMPLE III:
Under this example a method for contained (solid structure tank) retting involving the
microbial formulation with biofilm forming capability of the invention was carried out as
detailed hereunder:-
14

For such purpose a retting tank such as the one illustrated in accompanying figure 1 was
used. Such primary retting tank used is of 7 x 6 x 4 dimension and capable of holding more
than about 6000 I water. The tank can be made of cement and bricks. The inner wall of the
tank are preferably equipped with small circular rings through which ropes can be attached.
The primary tank is connected with effluent treatment/ composte tank through ports that
may be opened when necessary. The effluent treatment tank contains a trickling filter
system using a bed of rocks and sand. Treated effluent from this tank is accumulated into a
large tank/reservoir where the treated water may be stored. Appropriate cover of the tank
is preferred during the period of rainfall.
The method of carrying out the contained (solid structure tank) retting using the microbial
formulation of Example I above was as detailed hereunder :-
i) Jute stems were cut, leaves removed and the base of the stems submerged in
water for about 1 day;
ii) the stems were then spread on a flat surface;
iii) the bacterial inoculum concentrate was then diluted 1 : 100 in water before use;
iv) the diluted inoculum was next sprayed on the jute stems using automated or
manual spraying device whereby 1 litre of the concentrated solution was used for
3000 to about 6000 jute stems;
v) the stems were next air dried for 30 minutes and assembled in loose bunches of
about 50 each;
vi) the bunches were next placed on top of each other. The upper bunches were tied
down to rings attached. The tank was next filled with enough water to keep the
stems submerged (approximately 3000 liters - 6000 liters);
vii) that after 5 days approximately 2000 liters - 4000 liters water was released from
tank and fresh water added to earlier levels
15

viii) after this period, the pH of the system was adjusted to 7.0 with addition of about
2-3 kg Sodium Bicarbonate (common soda). The water was then agitated
vigorously;
ix) thereafter the system was again incubated till completion of retting;
x) thereafter completion of the retting, the stems are released and mechanically
moved to the next tank. Adequate amount of fresh water was added and the
stems washed to release the fiber. The washed fibers are kept for drying;
xi) following the washing, the corner vents of the primary tank were opened. This
allowed the sludge and spent liquor from the primary tank into the effluent
treatment tank. The empty primary tank was next kept ready immediately for a
second load.
xii) the jute leaves and twigs stored for compost formation were next placed into the
effluent treatment tank and mixed well with the sludge and optionally followed
with the addition of 2Kg of DAP. The system was incubated for about 3 days with
occasional stirring.
xiii) At the end of about 3 days the central vents were opened. This allowed clarified
water from the effluent treatment tank to enter the primary tank. This water was
ready to be recycled. Whenever the trickling filter partition clogged, it was
mechanically cleaned. The contents of the tank were vigorously mixed from time
to time.
xiv) The content of the primary tank was kept for processing for a period of 10-14
days, during which the most of the water was recycled. The compost was now
taken out and dried in the sun. The latter was packaged and ready to be
commercially sold or used as organic fertilizer.
16

EXAMPLE IV :
The process of retting carried out involving the microbial formaulation of the invention as
per Example III above and the product specifications obtained therefrom were tested and
compared to conventional retting process and product specifications and the results are
provided hereunder in Table II:
TABLE II

Jute
species Fiber
code Retting
conditions* Tensile
strength Fineness Grade Days
For
Retting
JRC 321 BREF
16 Example III 24.0 1.3 W2+40% 7
JRC 321 BREF
Z2 Control 28.7 1.4 W3+60% 21
As would be clearly apparent from the above results the process of retting of jute involving
the microbial biofilm forming formulation of the invention is much faster and results in good
quality fibre generation.
Reference is now invited to accompanying Figure 1 which illustrates an embodiment of the
system for jute fibre retting in accordance with the invention.
As shown in the figure the system basically includes a retting tank (RT),a trickling filter
tank (TFT) and a water recycling tank (WRT),As further illustrated in the figure said retting
tank is provided to favour the desired retting of the fibres under action of microbial
consortium /enzymatic treatment in accordance with the invention.
A communication means for supplying the effluent from the retting tank to said trickling
filter tank for treatment of the effluent under trickling filter technique is provided whereby
the organic waste from the bio process is converted to compost fertilizer.
17

The trickling filter tank is kept operatively connected to said water recycling tank such that
the water from the trickling filter tank could be recycled into the retting tank for
subsequent cycles of retting.
Advantageously, in the retting tank means preferably rings (RG) are provided to favour
securely tying the bundle of stems for retting with strings for the purpose of the retting.
The trickling filter tank is preferably comprised of plurality of filter media preferably
comprising a top large sized stone chip filter media comprising preferably 6 inch stone
chips, an intermediate sand filter media and a bottom smaller sized stone chip media
comprising preferably 4 inch stone chips.
The retting tank is operatively connected to the filter tank through an effluent transfer port
(ETP) at the top while the filter tank is operatively connected to the water recycling tank by
water transfer ports (WTP) near the base.
The above discussed system for retting is found to be simple and user friendly apart from
facilitating the faster and eco-friendly retting of jute fibres and the like.
Thus the present invention is directed to favour faster and cost-effective and uniform retting
for wide scale rural as well as industrial application of the invention in retting of bast fibres
such as jute. Moreover, as further apparent from the disclosure herein before the biofilm
forming microbial formulation for retting used in the present invention favours Potent
Extracellular Polymeric Substance (EPS) forming ability, allowing the (bacterial) components
of the invention to attach directly to the target stems (as biofilm), which in turn benefits
the retting process by (a) being naturally more tolerant/resistant to dynamic and harsh
(biochemical) process conditions than planktonic inoculum(s),(b) being not 'diluted' or
'washed away' in large water bodies (the latter being a fundamental process requisite) or
accidental flooding/rain and (c ) favouring better colonizing efficiency on the target stems,
ensuing better competition/survival against other associated microbes. Moreover, unlike
fungal enzymes which are active at low pH, the biofilm derived enzymes in the invented
bioprocess exhibit neutral pH optima. The bioprocess under the present invention can thus
be conducted at more environment friendly, neutral pH conditions than other inventions.
Importantly, the selective combination of the selective microbial/enzymatic formulation
18

19
further favour generation of the biofilm deliberately incorporated with more pectinase
producers, less cellulase/xylanases producers and no laccase produce so as to efficiently
degrade stem cementing material but not the embedded lignocellulosic fibers. No such
reported protocols have used this strategy. Also, considering that the rate of achieving jute
retting involving the formulation/enzymatic combination of the invention is much faster as
compared to presently available processes of retting, the retting of the present invention
would be user-friendly, eco-friendly, cost-effective and consistent for wide scale
application and use in retting and allied applications and production of such retted fibres for
variety of applications and end uses thereof.

Thus according to the basic aspect of the present invention there is provided a microbial
formulation providing for biofilm production useful for retting of bast fibres such as jute comprising:
biological bacterial constituents selected from one Acinetobacter sp., one Pantoea sp., one
Pseudomonas sp. and two Bacillus sp.in bacterial biofilm generating reagents and inoculum
media favouring Extracellular Polymeric Substance (EPS) forming ability.
In a preferred embodiment, biological constituents are selected from five bacterial species
i.e. a species of Acinetobacter sp., Pantoea agglomerans, , Bacillus pumilus, Bacillus subtilis
and Pseudomonas aeruginosa
In accordance to a preferred aspect, the microbial formulation providing for biofilm
production useful for retting of bast fibres is a bacterial consortium comprising a selective
combination of Acinetobacter sp., Pantoea agglomerans, and Bacillus pumilus. in bacterial
biofilm generating reagents and inoculum media favouring Extracellular Polymeric
Substance (EPS) forming ability.
Importantly, in the above microbial formulation the said bacterial constituents and inoculum
media are selectively provided such that the microbial biofilm produces high levels of
pectinases, low levels of cellulases and xylanses but no laccases.
In accordance with an aspect of the present invention the said bacterial bioflim generating
inoculum comprises a first reagent selected from Disodium hydrogen phosphate (Na2HPO4),
Potassium dihydrogen phosphate (KH2PO4), Sodium Chloride (NaCl) and Ammonium
Chloride (NH4Cl); and a second reagent selected from Pectin and nutrient broth.
In accordance with another aspect of the invention , the said microbial formulation includes
selective biological bacteria constituent„selected from one Acinetobacter sp., one Pantoea
sp., one Pseudomonas sp. and two Bacillus sp., all of which adopts the microaerophilic
growth mode in the low oxygen tension to synergistically favour the retting micro-
environment.
More specifically, according to a preferred aspect the biological bacterial constituents
comprise three species, Acinetobacter sp., Pantoea agglomerans, and Bacillus pumilus
selected from the above mentioned five bacterial constituents, which also favour the
microaerophilic growth mode in the low oxygen tension to synergistically favour retting
micro-environment
Advantageously, in the microbial formulation of the invention the said inoculum media
comprises sterilized reagents with pH of 7.0 i.e. neutral pH, in the substrate media.
Preferably, the microbial consortium is obtained as a concentrate suitable for dilution with water before use.
In accordance with another aspect of the invention the method for the manufacture of the
microbial formulation of the invention comprises:
dissolving pectin in water and adjusting the pH to 7.0;
introducing the substrate media;
adding sterilized portions of said first and second reagents;
adding bacterial constituents and subjecting to incubation to thereby provide the microbial
consortium in solution for retting of cellulose fibres.
Preferably, the said step of incubation is carried out at a temperature of 32 °C to 37 °C for a
period of about 3 days.
In accordance with another aspect of the invention there is provided a process for the
biochemical retting of bast fibres such as jute using the microbial formulation of the
invention which comprises: