(Deemed to be university)

TITLE OF THE PROJECT

Shake flask study for secondary metabolite production by Streptomyces spp.

A PROJECT SUBMITTED TO DR. D.Y. PATIL VIDYAPEETH (DEEMED TO BE

UNIVERSITY) IN PARTIAL FULFILLMENT OF FOUR YEARS FULL-TIME DEGREE
PROGRAMME B. TECH. (MEDICAL) BIOTECHNOLOGY

SUBMITTED BY
Yashi Gupta

UNDER THE GUIDANCE OF

Dr. Manmeet Ahuja

(Address of the host Institute)

IPCA Laboratories Ltd., Charkop Rd, Kandivali, Charkop, Charkop Industrial Estate,
Kandivali West, Mumbai, Maharashtra 400067.

DR.D.Y.PATIL BIOTECHNOLOGY & BIOINFORMATICS

INSTITUTE, TATHAWADE, PUNE – 33
(June 2021)

1
 CERTIFICATE
This is to certify that Ms. Yashi Gupta has prepared this project
titled “Shake flask study for secondary metabolite production
by Streptomyces spp.”, under my guidance and to my satisfaction,
in fulfillment of the requirement for B. Tech. degree in Medical
Biotechnology.

Signature & Seal of Guide

Guided By
Dr. Manmeet Ahuja.

(Address of the host Institute)

IPCA Laboratories Ltd. 125, Kandivali Industrial Estate, Kandivali

(West), Mumbai, Maharashtra 400067.
Director
Dr. D. Y. Patil Biotechnology & Bioinformatics Institute,
Tathawade, Pune – 33
(Deemed to be university)

2
 DECLARATION

I hereby declare this project titled “Shake flask study for secondary metabolite
production by Streptomyces spp.”, submitted to Dr. D.Y. Patil Vidyapeeth is my
individual work carried out at IPCA Laboratories Ltd., Mumbai under the guidance of Dr.
Manmeet Ahuja, for the fulfillment of the degree B. Tech Medical Biotechnology and
this project report or part has thereof has not been submitted elsewhere for any other
degree. I also undertake that the material reproduced in this thesis from other sources has
been duly acknowledged.

Place: Pune
Date: 10.06.2021 Yashi Gupta.

3
PLAGIARISM CERTIFICATION.

Plagiarism percentage is 8%.

4
 PREFACE
This dissertation is submitted in the partial fulfillment of the requirements for a Bachelor’s
degree in Medical Biotechnology at Dr. D.Y. Patil Biotechnology and Bioinformatics
Institute, Pune.

5
 ACKNOWLEDGMENT.

I would like to thank all my seniors who supported me during my dissertation project at
IPCA Laboratories Ltd. Biotech R & D, Mumbai. Foremost, I would like to express my
sincere gratitude to Dr. Devasis Guha, Senior General Manager of Biotech R & D, for
graciously accepting me for dissertation work. My special and huge thanks to the project
guide Dr. Manmeet Ahuja, whose enthusiasm and encouragement has been relentless
during my time as a student. Your guidance and advice have been crucial, not only to the
preparation of this thesis, but also to my confidence and professional development.

A huge thank you to the entire project team Vrushali Bhosle, Sejal Vora, Yashwant
Diwan, Shraddha Talele, Pooja Kain, Archana Tripathi and the Lab Technicians for
making such a good and vibrant place to work. I am so grateful to all for their loyalty,
kindness and good gesture.

Last, but not the least, I am greatly indebted to my family for their unconditional love,
care, support and blessings and to my friends who have been a great inspiration and support
throughout these four year and without them my graduation journey wouldn’t be that great
and joyful.

6
 ABBREVIATIONS.

❖ PMV- Packed Mycelial Volume.

❖ ISP-2- The International Streptomyces Project.
❖ APIs- Active Pharmaceutical Ingredients.
❖ YE- Yeast Extract.
❖ ME- Malt Extract.
❖ CSP- Corn Steep Powder.
❖ PEG- Poly Ethylene Glycol.

7
 CONTENTS.

1. Introduction-
1.1. History.
1.2. Operations.
1.3. Awards & recognitions.
1.4. Affiliations.

2. Details of the task performed-

2.1. Aim & objective.
2.2. Introduction-
2.2.1. Shake flask method.
2.2.2. Secondary metabolite.
2.3. Materials-
2.3.1. Media & reagents.
2.3.2. Equipments.
2.3.3. Instruments.
2.4.Methodology-
2.4.1. Microorganism & maintenance.
2.4.2. Shake flask method-
2.4.2.1.Development of the lab culture.
2.4.2.2.Development of the seed media & transfer of grown lab culture
into seed media.
2.4.2.3. Development of production media & transfer of grown seed
culture into production media.
2.4.2.4.Physical parameters determination-
2.4.2.4.1. pH of culture broth.
2.4.2.4.2. Packed mycelial volume (PMV).
2.4.2.4.3. Microscopy (Gram staining).
2.4.2.5. Analysis of secondary metabolite in broth sample through
HPLC.
2.4.2.6. Well plate method.

8
 2.5. Results.
2.5.1. Lab profiling.
2.5.2. Seed profiling.
2.5.3. Production profiling.
2.5.4. Well plate method.

3. Review of the task performed.

3.1.Critical analysis of the task performed.
3.2.Challenges faced & what measures were taken or could have been taken to
enhance the output of the said protocol.

4. Bibliography.

9
 1. Introduction.

IPCA Laboratories Limited is mumbai based international pharmaceutical

company. Its production includes theobromine, acetyl-thiophene, and P-Bromo
Toluene as APIs. It has produced 100+ formulations which includes tablets,
capsules, etc. The different variety of intermediaty drugs IPCA produces include
theobromine, acetyl-thiophene, and P- bromo toluene, distributing them in around
36 countries of Asia, Africa & South America.[1]

1.1.History.

IPCA Laboratories Limited (IPCA) was incorporated in 1949 in the name of 'Indian
Pharmaceutical Combine Association Ltd.' The company's name was converted to
IPCA Laboratories Ltd. on 6th August, 1964 and it was again renamed to IPCA
Laboratories Private Ltd' on 13th January, 1966. The current management had taken
over the company in November, 1975. IPCA's 1st APIs Plant for the manufacturing
of Chloroquine Phosphate was set up at Ratlam in the year 1986. In the year 1988,
the company acquired Hoechst India's formulations unit at Kandla.[1]

1.2.Operations.

IPCA is an integrated pharmaceutical company that manufactures as well as markets

more than 300 formulations and more than 80 API's that covers various therapeutic
sectors. IPCA has 10+ manufacturing units in India that manufactures API's and
formulations for the world market. IPCA's Formulations & APIs are approved by
all the major western regulatory authorities- FDA, MHRA, MCC, ANVISA and
TGA. IPCA manufactures APIs' such as modern equipments to update & tighten
R&D facilities. The company aims to develop newer drug delivery systems and
formulations for developed market and bio-equivalence studies of the same.[1]

10
1.1.Awards & recognitions.

In 2004, Forbes selected IPCA amongst the 200 'Best under a Billion Company' in
Asia. It also got certified from FDA, MHRA, MCC, ANVISA and TGA.[1]

11
 2. Details of the task performed.

2.1. Aims & objectives.

Aim: Shake flask study for secondary metabolite production by Streptomyces spp.
Objective: The objective of this project is to study the growth pattern of
Streptomyces spp. and to check for secondary metabolite production.

2.2. Introduction.

• Classical fermentation is majorly used for producing biologically important,

economically & environmentally friendly products.
• This process has been in use for ages for producing biologically important,
economically & environmentally friendly products.
• Actinomycetes are a group of bacteria which has an important part in the
production of variety of secondary metabolites which are known to be
biologically active compounds.
• Streptomyces is the largest group of genus of Actinobacteria harbour that have
the capability to produce a number of bioactive secondary metabolites, such as
the crucial therapeutic varieties of antibiotics, anti-cancer compounds,
antifungal and immunosuppressants.
• These are present mostly in soil & produce spores, & they have "earthy" smell,
which is produced from production of a metabolite, geosmin.
• Streptomyces have a complex secondary metabolism.
• Streptomyces make 80% of the clinically useful antibiotics of natural origin.
• The antibiotic name streptomycin is derived from Streptomyces. Streptomycetes
are pathogens, causing infections in humans.
• Streptomyces are Gram-positive, filamentous, aerobic bacteira, which produce
well-developed vegetative hyphae with branches.
• Streptomyces form a complex substrate mycelium that assists in scavenging
organic compounds from their substrates.[2][3]

12
Shake flask method-
• Medium enhancement by the process of shake flask technique is the most crucial
step which is done prior any step up of fermentation process.
• Hence, for forming an effective production media, fermentation variables like,
agitation speed, temperature, pH, dissolved oxygen & the right medium
components must be identified and optimized.
• An increased production makes the process cost tough that is required in the
changing market scenario.[2][3]

2.2.1. Secondary metabolite-

• Secondary metabolites are a different organic substance produced by bacteria,
fungi, plants and animals.
• They are molecules that are responsible for the overall homeostasis of the
organism.
• Secondary metabolites specifically modifies excretion of waste and toxic
products from the body.
• Fungus releases a variety of secondary metabolites during active cell growth.
• The secondary metabolites released by fungi have industrial usage.
• Hence, identifying these substances and enhancement of the fungal growth
conditions help us manage ample production of secondary metabolites.[4][5]

2.3. Materials-
2.3.1. Media and reagents-

• M1 media (merck).
• M2 media (merck).

• Lab media (HiMedia).

• Seed media (HiMedia).
• Production media (HiMedia).
• Antifungal powder.

• 50% glycerol solution.

• Acetonitrile.

13
 • Normal saline.
• Crystal violet.
• Gram’s iodine.

• Decolorizer.
• Saffranin.

2.3.2. Equipments-

• Petri plates (90 mm).

• Conical flask (250 ml).

• Volumetric flask (100ml).
• Slant tubes.
• Cryovials.

• Glass slides.
• Micropipette.
• Tip box.
• Pipette.

• Pipette gun.
• Centrifuge tubes.
• Wash bottles.
• Parafilm.

2.3.3. Instruments-

• Sonicator bath (Oscar).

• Centrifuge (Thermo lab).
• pH meter (Thermo scientific).
• Weight balance (Thermo lab).

• Magnetic stirrer (Remi).

• Incubator (Orbitech).
• Shaker incubater (Orbitech).

14
 • Compound microscope (Zeiss).
• -80°C Deep fridge (Thermo scientific).
• Biosafety cabinet (Esco).

2.4. METHODOLOGY.

2.4.1. Microorganism and maintenance-

• In this study, Streptomyces was used for the production of secondary

metabolite.

• The bacteria were grown on ISP-2 medium (composition mentioned

below).

• The culture was stored in incubator at 28 °C for 2 weeks.

• Streptomyces colony was grown in ISP-2 media and after 24 hrs. stored in
cryovials at -80°C (20ml culture broth + 18ml 50% glycerol solution).

2.4.2. Shake flask process-

2.4.2.1.Development of lab culture-

• ISP-2 media was selected from various literatures and was prepared as per
the composition given below and Streptomyces colony was maintained on
ISP-2 agar.

• ISP-2 media was inoculated with 1ml of culture from cryovial and growth
profiling was done for 34 hours at 240 rpm at 28°C in shaker incubator.

• Various parameters such as pH, PMV and microscopy (Gram staining) was
done.

• Growth profiling was done at 4 hrs. interval from 0th hr. to 16th hr. and
then profiling was done at 2 hrs. interval from 18th hr. to 34th hr.

• Various parameters such as pH, PMV and microscopy (Gram staining) was
done.
• Growth was seen at 22±2 hours and 0.3ml, 0.5ml & 1ml of ISP-2 was
transferred each to S1, S2 & S3.

15
 Inoculated lab media
0.3ml 0.5ml 1ml

S1, S2, S3 S1, S2, S3 S1, S2, S3

Sr. No. Composition (gram/litre) M

1 YE 4
2 ME 10
3 D-glucose monohydrate 4
pH adjusted to 7.0

2.4.2.2.Development of seed media and transfer of grown lab culture into seed
media-

• S1, S2 & S3 media were selected from different literatures and 1% (0.3ml),
1.5% (0.5ml) & 3% (1ml) of matured lab inoculum was transferred into
each 30 ml S1, S2 and S3 seed media in 250 ml volumetric flask.

• These flasks were stored at 28 °C at 240 rpm in shaker incubator for 30 hrs.

• Various parameters such as pH, PMV and microscopy (Gram staining) was
done.

• Growth was checked at 26 hrs. and 30 hrs. and growth was finalised at 26
hrs.

• Out of the 3 medias with different concentrations, S3 media with the lab
culture concentration of 1% (0.3 ml) showed good growth while others
showed poor growth.

• 1% of lab culture was again inoculated in 30 ml of S3 in 250ml flask and

final profiling was done for 47 hrs.

• Various parameters such as pH, PMV and microscopy (Gram staining) was
done.

16
 • Growth profiling was done at 4 hrs. interval from 0th hr. to 20th hr.,
followed by 3 hrs. interval from 23rd hr. to 35th hr. and finally 2 hrs.
interval from 37th hr. to 47th hr.

• Growth was seen at 24±2 hrs. and 3ml of seed culture was transferred to
production media.

Sr. No. Composition (g/L) S3

1 Soluble starch 20.0
2 Dextrose -
3 Dextrin White -
4 Milk powder -
5 Cotton seed meal -
6 Tryptone soy broth 20.0
7 MgSO4 0.025
8 FeSO4 -
9 NaCl -
10 CaCl2 -
11 ZnSO4 -
12 MnSO4 -
13 CaCO3 3.0
14 K2HPO4 1.0
15 Phosphate buffer pH 6.8* -
16 Corn starch -
17 Soya flour -
18 Inactive dry yeast -
19 YE 3.0
pH adjusted to 7.2

*phosphate buffer saline (g/100mL)-Di-sodium hydrogen phosphate-2.82,

potassium dihydrogen phosphate-1.14. Autoclave for 15 mins.

17
2.4.2.3.Development of production media and transfer of grown seed culture
into production media-
• 6 production medias were selected from various literatures on the basis of
carbon, nitrogen, oxygen, sulphur and various other sources and 3ml of
grown seed culture (S3-0.3ml) was inoculated in 30ml of production
medias in 250ml flask.

• Growth profiling of P1-P6 was done for 10 days (240hrs.) at 24 hrs. interval
at 240rpm at 28°C in shaker incubator and various parameters such as pH,
PMV and microscopy (Gram staining) was done.

• Out of P1-P6, P3 and P6 showed good growth while P1, P2, P4, P5 showed
poor growth.

• P3 & P6 were selected as final production media and final profiling of P3

& P6 was done.

S. No. Composition (g/L) P3 P6

1 Dextrose 5.0 -
2 Soluble starch - -
3 Beef extract - -
4 Bacteriological peptone - -
5 Sodium chloride - -
6 Soya flour 10.0 10.0
7 Ebios (active yeast) - -
8 Magnesium chloride - -
9 Copper sulphate - -
10 Zinc sulphate - -
11 Calcium carbonate 1.50 1.50
12 Dextrin white 90.0 140.0
13 Soya peptone 10.0 10.0
14 Glycerol 10.0 10.0
15 L-Lysine 2.50 -

18
 16 K2HPO4 1.0 0.8
17 PEG 1.0 12.5
18 CSP - -
19 KH2PO4 - -
20 (NH4)2SO4 - -
21 Magnesium sulphate - -
22 Yeast extract powder - -
23 Soya oil - -
24 Cotton seed meal - 14.0
pH adjusted to 7.0 7.2

2.4.2.4.Physical parameter estimation-

The variables such as PMV, pH, and microscopic appearances were
calculated at variable time frames.

2.4.2.4.1. pH of culture broth-

• pH meter is maintained at constant with neutral, acidic and basic buffer.
• pH electrode is washed with demineralised H2O and cleaned it with tissue
wipes.

• Dip the electrode rod into the sample and measure the stable pH value.

pH reading of a media using ph meter.

19
2.4.2.4.2. Packed mycelial volume (PMV)-
• The cell volume of the fermentation broth is calculated by dispensing 10 ml
of broth sample in 15 ml centrifuge tube and is spun at 4500 rpm for a
duration of 5 mins.

• The formula for PMV calculation is given below-

PMV determination using centrifuge maching with swinging bucket rotor.

2.4.2.4.3. Microscopy-
• The sample was spread on a clean slide in order to make a smear.
• The smear was fixed onto the slide by fixing the sample by heating it on a
burner. Gram staining was done and was observed under the microscope
(100X-oil immersion).

20
 Inverted microscope with camera.

2.4.2.5.Analysis of secondary metabolite in broth samples through HPLC-

• Acetonitrile was used for the extraction of fermentation broth.

• 5 gm sample (P3-168h, 192h, 216h, 240 & P6- 168h, 192h, 216h, 240) was
weighed and dissolved in 10 ml of acetonitrile and was sonicated for 15
mins and filtered.

• A total of 4 extracts were obtained each for P3 & P6.

2.4.2.6.Well plate method-

• ISP-2 agar slants were made and 0.2 ml of culture suspension of C.albicans
was spread onto the agar and was incubated at 28°C for 2 days.

• 65gm of SDA agar was suspended in 1000ml of distilled water (2-3 spoons
of agar agar was added for faster solidification of SDA) and was autoclaved
for 20 mins at 121 atm pressure.

• Normal saline (0.85 gm of NaCl in 100 ml of water) was used for harvesting
C.albicans & 1 ml of it was suspended in SDA agar and rigorous shaking
was done to ensure that the suspension is equally distributed in the agar
media.

21
 • SDA+suspension was poured in a big petri plate and were left in biosafety
for solidification.

• 10 wells were made in plate and 4 samples of P3 (168H, 192H, 216H,

240H) and 4 samples of P6 (168H, 192H, 216H, 240H) were loaded as
follows-

• 100µL of the extracts, 100µL of acetonitrile & 100µL of antifungal powder

solution (pinch of powder in 500 µL of normal saline).

• The plate was incubated at 28°C in incubator for 10 days and after 10 days
antifungal resistance was to be observed.

2.5. RESULTS.
2.5.1. Lab profiling.
Finalised lab profiling- growth seen at 22 H.
Sr. No: Age (H) pH PMV (%)
1 0 6.70 0
2 4 6.83 1
3 8 6.84 2
4 12 7.22 4
5 16 6.92 6
6 18 6.82 8
7 20 5.87 8
8 22 5.71 8
9 24 6.04 8
10 26 5.98 6
11 28 5.81 4
12 30 5.85 3
13 32 5.82 2
14 34 6.04 1

22
Microscopy- Gram staining (age in hrs.)-

4 8

12 16

18 20

22 24

23
26 28

30 32

34

24
Flasks and tubes.
12HRS.

9 8
8
7.5
7
6 7
PMV (%)

PH
6.5
4
3 6
2
5.5
1
0 5
0 4 8 12 16 18 20 22 24 26 28 30 32 34
AGE (H)

Growth profiling of lab media.

25
2.5.2. Seed profiling.
Finalised seed profiling (S3-0.3ml)- growth seen at 26 H.

Sr. No: Age(H) pH PMV (%)

1 0 7.45 2
2 4 7.53 3
3 8 7.88 4
4 12 7.95 5
5 16 7.33 6
6 20 6.95 7
7 23 6.60 9
8 26 6.55 10
9 29 6.62 10
10 32 7.64 10
11 35 7.83 10
12 37 8.17 10
13 39 8.26 10
14 41 8.30 10
15 43 8.42 8
16 45 8.41 7
17 47 8.46 6

Microscopy- Gram staining (age in hrs.)-

4 8

26
 12 16

20 23

26 29

32 35

27
37 39

41 43

45 47

28
Flasks and PMV tubes.
12 HRS.

26 HRS.

29
 Chart Title
9
14
8
12 7
10 6
PMV (%) 5
8

PH
4
6
3
4
2
2 1
0 0
0 4 8 12 16 20 23 26 29 32 35 37 39 41 43 45 47
AGE (H)

Growth profiling of S3 0.3ml

1.1.1. Production profiling

Finalised production profiling (P3 & P6-3ml).

Samples Sr. No: Age (H) pH PMV (%)

1 0 6.47 4
2 24 6.13 4
3 48 6.76 5
4 72 5.57 7
5 96 5.70 20
P3 6 120 5.42 22
7 144 5.54 22
8 168 5.79 22
9 192 6.40 22
10 216 6.96 22
11 240 6.94 22

1 0 6.70 9
2 24 6.93 10
3 48 6.74 11
4 72 6.80 15

30
 5 96 6.63 22
P6 6 120 5.79 25
7 144 5.65 25
8 168 5.66 25
9 192 6.22 25
10 216 8.23 25
11 240 6.26 25

Microscopy- Gram staining (in hrs.)-

P3.

24 48

72 96

120 144

31
 168 192

216 240
Flasks and PMV tubes.
P3.
144 HRS.

32
168 HRS.

192 HRS.

33
216 HRS.

240 HRS.

34
 25 8
7
20
6

15 5

PMV (%)

PH
4
10 3
2
5
1
0 0
0 24 48 72 96 120 144 168 192 216 240
AGE (H)

Growth profiling of P3.

P6 (age in hrs.)-

24 48

72 96

120 144

35
 168 192

216 240
Flasks and PMV tubes
P6
144 HRS.

36
168 HRS.

192 HRS.

37
216 HRS.

240 HRS.

38
 Chart Title

35 9

30 8.5
8
25
7.5
20
PMV

PH
7
15
6.5
10
6
5 5.5
0 5
0 24 48 72 96 120 144 168 192 216 240
AGE (H) PMV (%) pH

Growth profiling of P6.

Well plate method.

Zone of inhibition

Zone of inhibition of Streptomyces against C.albicans.

39
 3. Review of the task performed.

3.1.Critical analysis of the work carried out.

• To boost the production in shake flask studies, two different approaches were
observed.

• At 1st, “Two stage Fermentation” approach was put in use in which grown lab
inoculum was directly transferred in 6 production medias out of which 2 medias
were selected for the production stage.

• The second method consisted of “Three stage Fermentation” method in which

grown lab inoculum was transferred into seed media and in turn grown seed culture
was transferred into production media.

• The seed culture’s age was monitored for the improvement of the yield. The
maximum concentration was achieved in the second approach i.e. “Three Stage
Fermentation”.

• Thus, it was decided that “Three stage Fermentation” on selected media was the
better option for the production of pharmaceutical product.

• In the 2nd method, secondary metabolite production was majorly affected by adding
one more seed stage.

• Considering the above methodology, herein three stage fermentation approach was
used.
• Media used in three different stages differs from one another: Lab Media is used in
1st stage, is composed of normal nutritional requirement for the culture.

• Generally, lab media is composed of component containing nitrogen source, carbon

source.

• Seed media is used in 2nd stage, contain component with good source of carbon,
nitrogen, minerals and buffering agent.

• Production media is used in 3rd stage is similar to seed media only have different
carbon and nitrogen source.

• The microbe used during the production of Stage 1 inoculum was Mycelium
Filamentous.

40
• This microbe was also isolated with the help of a particular predesigned strain to
that particular microbe.

• It was later transferred to the Seeding inoculum and was observed for 10-15days.
• After this, the microbe was transferred into a production inoculum batch and kept
for 10-15 days.

• The time difference observed here for many days is taken into account considering
not only the precise number of days it will take for the mycelium cells to germinate
and form multiple fibres like structure.

• Though the cells are transferred from the inoculum stage to seeding inoculum and
finally to production inoculum in a span of just 10days, the additional span of 2-4
days is taken into account on two grounds; Biochemical analysis period and any
holidays.

• During transmission of this period, the samples were daily checked for oncological
and contamination observing purposes.

• It was observed that the mycelium growth culture initially had just a couple of
filaments spread across in the inoculum when observed under a microscope.

• This, due to overtime multiple folds of multiplication eventually lead to the

formulation of multiple fibres clustering together.

• This eventually led to the formation of a dense pellet after being allowed to grow
for days and hours together.

• The morphological characteristics also helped determine the stages of development

of the microbe.

• During stage 2 seeding batch, when the microbe was inoculated, it has a very less
amount of fibrous content around the culture.

• In stage 3 production batch, these culture filaments further condensed together in

order to form a pellet.

• With growing number of hours (up to 240 hrs range) it was observed under an
optical microscope (100x for Gram Staining) that the pellets were very dense and
accumulated altogether.

41
• The right harvesting time for the broth was not only determined using various other
chemical parameters like pH, PMV, but also considering the microbial morphology
of Mycelia filamentous.

• Around the dying age of the microbe, we can observe minor breaks in the filament
strands of the microbe itself.

• Hence, determining the right time to harvest as the microbes would no longer grow
and produce the desired pharmaceutical product due to its cell degradation.

• When filamentous organisms are grown in submerged culture, they showcase

different morphological forms. These forms range between scattered single
mycelial fragments and thick tangled mycelial masses that are known as pellets.
• Gradually, the mycelial volume increases with age that results in thick meshwork
of hyphae.
• The hyphael branches seen were smooth and thick at an early age. It later became
thin and dense.
• The study presented herein, “Three stage fermentation” method was put in use for
secondary metabolite production in shake flasks and then scaleup into fermenter.

• The method had three stages in which transference of grown lab into seed media &
from seed media to production medium occurred.

• Because of seed medium, increase in inoculum & least lethal effect of cells
happened.

• Some factors such as morphological structure & metabolic activity are critical
constituents for the proper manufacturing of bioactive metabolites.

• In shake flask study, shake flask is a classical parallel reactor used for fermenter.
• Prior to this, colony characterization and culture development is done, which
ultimately isolate, screen and prepares sporulating culture for seed profiling and
production.

• Seed profiling enhances the culture and prepares it for next production stage where
it releases secondary metabolites.

• After seed profiling, spores are transferred to the production stage, where it gives
the amount of metabolites produced.
• This forms the lab grown inoculum (STAGE I) which is pooled in the glass
assembly.

42
• Then the primary objective was achieved by scaling up the lab grown inoculum
into the fermenter.

• The fermenter comprises two stages : Seed (STAGE II) and Production (STAGE
III).

• The lab grown inoculum with the highest activity in shake flask is transferred into
the stage II and when it achieves a maximum PMV it is transferred into the stage
III.

• In stage III, various parameters were monitored.

• As per the above conducted experiment, it was observed how the batch was
moderated and determined on the basis of its biochemical physical, chemical in
process sample microscopic observation.

• In this batch, the bacteira had an optimum pH range of 5-6 pH during the productive
phase.

• In light of the above, we conclude that the said parameters enhances the activity to
derive optimum yield of the defined pharmaceutical product.

3.2.Challenges faced and what measures were taken or could have been taken
to enhance the output of the said protocol.
• There were issues in contamination of the media as Streptomyces gets easily
contaminated. So, proper protocols were followed to prevent the contamination of
the media.
• The growth curve was not proper at first, so the growth profiling was re-run to
obtain proper curve in terms of PMV.
• For the well plate method, the agar media with the pathogen was properly shaken
before pouring in the plate to ensure proper distribution of the pathogen in the agar
media.

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 4. Bibliography.
1. www.business-standard.com.
2. Umesh Luthra, Harish Kumar, Archana Tripathi, Shradha Talele, Vandana Gupte.
"Study on growth and ascomycin production by Streptomyces hygroscopicus
subsp. Ascomyceticus", Biocatalysis and Agricultural Biotechnology, 2019.
3. en.wikipedia.org.
4. R. Thilagam, N. Hemalatha. "Plant growth promotion and chilli anthracnose
disease suppression ability of rhizosphere soil actinobacteria", Journal of Applied
Microbiology, 2019.
5. Formulation and Statistical Optimization of Culture medium for Improved
Production of antimicrobial compound by Streptomyces sp. JAJ06.

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