UNIVERSITI PUTRA MALAYSIA

FACULTY OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES

SEMESTER 2 SESSION 2020/2021

BTC 3305: BIOPROCESSING AND BIOMANUFACTURING DESIGN

MINI PROJECT REPORT: PRODUCTION OF HYALURONIC ACID BY FOOD

GRADE MICROORGANISM AS FOOD SUPPLEMENT

NAME MATRIC NUMBER

ANIS SHAZWINA BINTI SAHAK 196763

NABIHAH BINTI KU MOHAMMAD SOFFI 196946

NOR SYAKIRAH BINTI ZAINAL 196666

RAM KUMAAR A/L PILLAI 197002

PROGRAM: BACHELOR OF BIOTECHNOLOGY WITH HONOURS

LECTURER: 1) PROF DR HIDAYAH ARIFFIN

2) ASSOC. PROF DR MURNI HALIM

TABLE OF CONTENT
 CONTENTS PAGE NUMBER

EXECUTIVE SUMMARY

DESIGN OBJECTIVES

PROCESS DESCRIPTION

DATA BOOK

PROCESS ANALYSIS: BFD

PROCESS ANALYSIS: PFD

MASS BALANCE SHEET

TECHNOECONOMIC EVALUATION

PLANT LAYOUT

SAFETY MEASURE &

ENVIRONMENTAL IMPACT

CONCLUSION
1.0 EXECUTIVE SUMMARY

Hyaluronic acid (HA) is a hydrated gel and comprises repeating units of glucuronic
acid and N-acetylglucosamine. Production and recovery of HA has gained great importance
due to its vast clinical applications. Currently, large scale production of HA involves
extraction from animal tissues as well as the use of bacterial expression systems in
Streptococci. However, due to concerns over safety, alternative sources of HA have been
pursued which include the use of endotoxin-free microorganisms. HA production through
fermentation by generally recognized as safe (GRAS) microbial strains emerged as an
attractive alternative. However, to ensure the physicochemical and biological properties, a
purity near to 99 % is a primary requirement, aiming for clinical applications. To achieve this
goal, various downstream operations have been used, aiming for HA concentration,
separation and purification.
In pursuit of obtaining highly pure HA, we have developed a fed-batch fermentation
process using Streptococcus thermophilus in a 35 000 L stirred tank bioreactor that resulted
in a maximum yield of 2.408 g/L HA. In addition, an efficient method for separation and
recovery of hyaluronic acid from a highly viscous broth are done by treating with
trichloroacetic acid (0.1%) and charcoal (1-2%), passing through filtration (0.45 μm) and
ultrafiltration that resulted in recovery of 70 % of clinical grade HA with molecular weight of
2.5×106 Da. These process resulted in higher capital and operating costs, but also in higher
profits, because HA for injectable use has a higher selling price that more than compensates
for its higher production costs.

2.0 OBJECTIVES

2.1 GENERAL OBJECTIVES

To produce food grade microbial hyaluronic acid to be used as food supplements

2.2 DESIGN OBJECTIVES

● To produce 99% purity food grade hyaluronic acid from Streptococcus

thermophilus to be used as a food supplement.

● To produce 6000 kg of food grade hyaluronic acid from Streptococcus

thermophilus annually to meet 1% world demand.

3.0 PROCESS DESCRIPTION

3.1 BACKGROUND OF PRODUCT

Product Microbial food grade hyaluronic acid

Function Food supplement

Target user People with osteoarthritis

Form Solid (Tablet)

Administration Oral

% of purity 99% of Hyaluronic acid

3.2 BACKGROUND OF PROCESS

DESIGN BASIS

Parameter Specification

Production scale 10 MT

Annual operating time 2160 hours (90 days)

Process cycle time 96 hours

Concentration of HA per batch 2.408 g/L

Amount of HA produced per batch 66.46 kg

% Percent purity of HA produced 99%

% Impurities in final product <0.1%

FERMENTATION CONDITION

Parameter Condition

Mode of fermentation Fed-batch

Temperature 40℃

pH 6.8

Duration 24 h
 Agitation speed 230 rpm

Medium Carbon source: Skimmed milk

Nitrogen source: Yeast extract

PROCESS UNIT (RAM)

Steps Description

1) Inoculum Preparation Strain used: Streptococcus thermophilus

(GRAS) obtained from Yakult Centre
Institute cultivated in de Man Rogosa and
Sharpe (MRS) medium

Inoculum development: Three seed

fermentation steps where each consecutive
fermenter expansion factor is of 10×

2) Fermentation Suitable environment for fermentation

3) Primary Recovery
Centrifugation: To separate the bacterial
cells from the HA molecules

Filtration using 0.22 μm: Removal any

residual cells

Ultrafiltration/Diafiltration

4) Purification Trichloroacetic acid (0.1%) and activated

charcoal (1-2%) followed by centrifugation.

Filtration: Ultrafiltration in diafiltration

mode.

5) Vacuum Drying
Vacuum dryers are very efficient in heat
sensitive
 Average drying temperature is very low than
standard dryers which able to save energy.

3.8 Problem and Constraints (syak)

1. Source of microorganism used, Streptococci is pathogenic thus not suitable for food

grade substance production due to the risk of hyaluronidase or exotoxin

contamination.

2. Amount of Hyaluronic Acid produce by Streptococcus thermophilus is low which is

10 mg/L only (Izawa et al., 2009)

3. Purification of Hyaluronic Acid is difficult and costly as it is complexed with

proteoglycans (Izawa et al., 2009).

4. The safety and regulation of food grade products must achieve at least 95% purity and

must contain GRAS status (Schieving, 2017).

Solutions

PROBLEM SOLUTION

Source of microorganism used , Used Streptococcus thermophilus YIT

 Streptococci is pathogenic thus not suitable 2084 obtained from Yakult Centre Institute
for food grade substance production due to instead of using Streptococcus
the risk of hyaluronidase or exotoxin zooepidemicus . This is due to
contamination. Streptococcus thermophilus YIT 2084 has
(GRAS) status and does not produce
exotoxin.

Amount of Hyaluronic Acid produce by Optimization of medium and fermentation

Streptococcus thermophilus is low which is conditions.
10 mg/L only (Izawa et al., 2009)

Purification of Hyaluronic Acid is difficult As Streptococcus thermophilus YIT 2084

and costly as it is complexed with has (GRAS) status and does not produce
proteoglycans (Izawa et al., 2009). exotoxin, precipitation with
tricholoroacetic acid and charcoal treatment
was chosen instead of isopropanol
precipitation. Besides that, used
ultrafiltration in diafiltration mode and
vacuum dried.

The safety and regulation of food grade Used precipitation with tricholoroacetic
product is it must achieve at least 95% acid and charcoal treatment, centrifugation,
purity and must contain GRAS status ultrafiltration in diafiltration mode and
(Schieving, 2017). vacuum dried to achieve 99% purity.

4.0 Data book of reactants, process and products (biha)

1. Products

● Uses:

Hyaluronic acid is a substance that is naturally present in the human body. It is found

in the highest concentrations in fluids in the eyes and joints. Hyaluronic acid is the
 product of interest that will be used in food supplement form. A 2015 study published

in Rheumatology International reported that a three-month course of an oral

hyaluronic supplement called Oralvisc offered relief to overweight adults with knee

osteoarthritis (Zonca et al., 2015). Oral intake of high purity hyaluronic acid is

effective in the treatment of American patients of knee osteoarthritis.

● Form

The hyaluronic acid produced will enter vacuum drier to convert the liquid form of

hyaluronic acid into powdered form of hyaluronic acid after undergoing a few

primary recovery and purification steps.

● Quality

The objective of the process design is to produce 99% purity of hyaluronic acid as it

will be commercialised as a food supplement to treat osteoarthritis. Thus,

approximately less than 1% of hyaluronic acid consists of impurities and other by-

products. This purity will contribute to the effectiveness of the hyaluronic acid on

treating consumer’s joint problems. Besides, the unit operations and processes are

proposed to produce high molecular weight of hyaluronic acid (1000kDa) in order to

maximize the benefits of the supplement in treating osteoarthritis.

2. Reactants

● Availability

Streptococcus thermophilus culture is obtained from culture collection and

bought in ampoules for every batch as annual subscription. Approximately 8

kg of the culture is used to develop inoculum.

● Impurities

The cell debris and unwanted protein are formed during the primary recovery

steps and all of the impurities will be subsequently removed throughout the
 proposed purification step until less than 1% impurities are present in the final

products.

3. Reactions

● Phase

The culture is used during its log phase

● Kinetics

Fed-batch fermentation.

● By-products

The fermentation of hyaluronic acid by Streptococcus Thermophilus lead to

the formation of many by-products which are acetic acid and lactic acid. These

by-products increase the viscosity of the fermentation product and require

many purification steps such as activated charcoal adsorption, dead-end

filtration and ultrafiltration.

4. Physical properties

Molar mass :3–7 x 106 g/mol

Empirical formula :(C14H21NO11)n

Appearance :Transparent, viscous fluid or white powder

Water solubility :Infinite

5. Chemical properties

HA is not hazardous and not toxic to humans, animals and the environment.

Degradation of HA leads to a decrease in the molecular weight and, consequently, to a

decrease in viscosity, which is detrimental to the use of HA.

Process synthesis: Synthesis Tree Diagram

5.0 Process Analysis: BFD (anis)

Figure 1. Block Flow Diagram (BFD) for production high molecular weight hyaluronic acid
to be used as food supplement using Streptococcus thermophilus

6.0 Process analysis: PFD (biha)

Figure 2. Process flow diagram (PFD) for production of high molecular weight of hyaluronic
acid to be used as food supplement using Streptococcus thermophilus

7.0 Mass Balance sheet (based on PFD)

The mass stoichiometry of this reaction is given below:

100 Lactose + 1 Salts + 165 Yeast Extract → 5.2 Acetic Acid + 18.6 Biomass + 80 Carbon

Dioxide +8.7 Hyaluronan + 27.5 Lactic Acid + 126 Water

Figure 3. Mass Balance sheet

8.0 Technoeconomic evaluation (biha/syak/ram)

8.1 Technical analysis:

8.1.1 The process

1) Efficiency

The main factor that disrupt efficiency of the HA production is contamination . Potential

contaminating peptides/proteins in the broth is 21 g/L per batch .Yeast extract (21.1 g/L), the
main source of contaminating peptides that must and will be eliminated in the downstream

portion of the process.

2) Number of steps

• Upstream Section (Mixing , Seed train , Fermentation )

• Primary Recovery Section (Centrifugation , Filtration , Ultrafiltration/Diafiltration)

• Purification Section (Trichloroacetic acetic+activated charcoal adsorption , centrifugation ,

Ultrafiltration/Diafiltration)

• Preservation ( Vacuum drying )

3) Productivity

Is defined as Output volume/ Input volume

Input volume includes :

1) Salts (1.2kg)

2) Skimmed milk (362.5kg)

3) Yeast Extract (906.3kg)

4) Water ( 3000L/3000kg)

Output volume:
1) Hyaluronic Acid (66.46 kg)

Productivity = 0.3981 seconds-1

8.1.2 The product characteristics produced through the proposed process

1) Degradation

Synthesis of a single HA dimer is clearly energy-intensive where it consumes 5 ATP

equivalents; 2 NAD+ molecules; 1 acetyl-CoA molecule and 1 glutamine molecule. In

addition , HA precursors come from G6P and F6P. Firstly , HA synthesis interfere with ATP

production (pentose-phosphate pathway). Secondly , HA synthesis also interfere with

production of cell wall components in bacteria because G1P, UDP-glucose , and UDP-GlcNA

are necessary to produce cell wall , teichnoic acids and peptidoglycan .In conclusion ,

technically , HA synthesis competes with ATP production and Biomass growth where higher

intracellular concentrations of key precursors like UDP-GlcNA , lead to higher Molecular

weights.

2) Product yield

-Primary recovery would produce a product yield of 96%. Purification , HA precipitation

yield will be assumed to be 95%. Final product yield will be 99% HA in powder form .

3) Stability
The culture and environment prepared in a way that the S.thermophilus could last for 24

hours under skimmed milk culture , 230 rpm , pH 6.8 ,40℃ temperature without mutation .

4) Important characteristics related to its application

HA’s properties are closely related to its molecular weight (MW), which typically ranges

from 103 to 107 Da. Due to its large MW and capacity to establish multiple hydrogen bonds

with water molecules, HA is very hygroscopic and exhibits unique rheological and adhesive

properties . At concentrations as low as 0.2%, HA solutions present highly non-Newtonian,

shear-thinning behavior, and at concentrations higher than 1.5%, HA forms stable hydrogels.

High-MW HA has lubrication, shock absorption, space-filling, anti-inflammatory and anti-

angiogenic properties, whereas low-MW HA is pro-inflammatory and susceptible for

hydration, wound healing, and space-filling properties . 0.5–4 MDa as a topical cream for

wound healing, 0.1–0.5 MDa and for the preparation of anti-wrinkle products.

EQUIPMENT REQUIRED

Table 1.1 The equipment required for the processes and its description.

RAW MATERIALS REQUIRED

Table 1.2 The total amount of raw materials required and the total cost per batch
Raw materials Amount Total cost Sources
(kg)/batch (RM)/batch

a 362.5 2639 https://www.alibaba.com/

Skimmed milk
product-detail/SKIMMED-
MILK-
POWDER_50022201649.html?
spm=a2700.galleryofferlist.norm
al_offer.d_image.1b3428c20a9Y
Lv

a 906.3 9534.28 https://www.alibaba.com/

Yeast extract
product-detail/Yeast-Extract-
Yeast-Yeast-Extract-
Yeast_60740128916.html?
spm=a2700.galleryofferlist.norm
al_offer.d_image.13b41837mXF
ubr&s=p

b 3 (5% royalty annually) https://www.phe-

Streptococcus
culturecollections.org.uk/
thermophilus products/bacteria/detail.jsp?
refId=NCTC+10353&collection
=nctc

a 27.6 580.70 https://www.alibaba.com/

Tricholoroacetic
product-detail/Trichloroacetic-
acid acid-TCA-CAS-76-
03_1600261323630.html?
spm=a2700.galleryofferlist.norm
al_offer.d_title.23b075e2yobO2
5

a 552 2323.92 https://www.alibaba.com/

Activated charcoal
product-detail/Activated-
Charcoal-High-Quality-China-
Organic_62003959617.html?
spm=a2700.7735675.normal_off
er.d_image.577d615cPcV2Xu&s
=p

a 25.2 158.76 https://www.alibaba.com/

Salts
product-detail/edible-salt-Salt-
 In-Best-
Quality_62006555171.html?
spm=a2700.galleryofferlist.norm
al_offer.d_title.78455d522XACc
w

c 24 m3 49.68
Water, H2O

Total cost per/batch (RM) 15 135.15

Total cost per/month (RM) 113 513.63

a
Prices are referred to Alibaba online trade show.

b
Prices are referred to Public Health England Culture Collection

c
is referred to the latest water tariff for commercial usage by Air Selangor (1-35 m3 : RM
2.07; 35 m3 and above: RM 2.28)

COST FOR ENERGY USAGE

Table 1.3 The total energy usage for all the equipment.

Table 1.4 The total overhead cost per month

Overhead cost Amount Total cost (RM)/month

Labour (Operator) 10 20000

*including SOCSO, KWSP (Rm2000 per 1 person)

Labour (Engineer) 6 36000

*including SOCSO, KWSP (Rm6000 per 1 person)

Electric bill 1407.6KWh/batch 5447.40

(RM726.32/bacth)

Raw material cost -

113 513.63

Total cost per month 174 961.03

Price is referred to electricity tariff by TNB (1-200 kWh: RM0.218; 201-300KwH: RM0.334,
301-600kWh:
RM0.516)
EQUIPMENT PURCHASE COST TABLE

Table 1.5 The total cost for all the equipment required.

FIXED CAPITAL INVESTMENT TABLE

Table 1.6 The total fixed capacity investment.

Process Cost (RM)

A. Total Plant Direct Cost (TPDC)

1. Equipment Purchase Cost (PC) 11 900 556

2. Installation 463911.99

3. Process piping 671191.81

4. Instrumentation 256632.20

13059
5. Electricals

6. Buildings (process, administration, 177668.42

maintenance)

7. Yard improvement 98704.68

8. Auxillary facilities 542875.73

Total (TPDC) 14 124 599.83

 B. Total Plant Indirect Cost (TPIC)

9. Engineering 695861.70

10. Construction 811838.65

Total (TPIC) 1507700.35

C. Total Plant Cost (TPDC + TPIC) 15 632 300.18

11. Contractor fee 229634.34

12. Contingency 306179.00

D. Direct Fixed Capital (DFC) TPC+11+12 16 168 113.52

LABOUR COST TABLE

Table 1.7 The labour requirement and cost summary

Section Amount of staff Monthly salary cost Annual salary cost

Operator 10 20000 240000

Engineer 6 36000 432000

Total 672000

RAW MATERIAL COST TABLE

Table 1.8 The total cost and amount for raw material required

ANNUAL OPERATING COST TABLE

Table 1.9 The summary of annual operating cost

Cost Item RM/year %

 Raw material 1 362 163.56 9.77

Labour 672000 4.82

Equipment 11 900 556 85.37

Transportation 6000 0.04

Total 13 940 719.56 100

PROFITABILITY ANALYSIS TABLE

Table 1.10 The analysis of profitability of the project.

9.0 Plant layout, safety measures and environmental impact

9.1 Plant layout (anis)

9.2 Safety Measures

Equipments Safety measures

1. Autoclaving

• Wearing appropriate Personal Protective Equipment (PPE)

including a lab coat, heat resistant gloves, and eye protection,
especially when unloading the autoclave.

• Never sealing containers; under pressure they pose an explosion

risk.

• Never opening the door to the autoclave if there is water running out
the bottom. Clogged steam lines, equipment malfunction, or plugged
drains may cause a buildup of scalding water

2. Piping

• Should not react with the active material or excipients used in the
pharmaceutical manufacturing
-SS 304 – outside production site
-SS 316 L – Inside the production site

• SS 316 L used in the production site as has direct contact with

product / non-magnetic and not hardenable by heat treatment.
3. Fermentation seed
tank
• Use only safe microorganisms from safe sources.

• Do not seal bioreactors but prevent the entry of external

microorganisms and the release of aerosols.

• Sterilise all equipment before and after use and sterilise the culture
before disposal.

• Keep electrical leads tidy and site mains equipment as far away
from the reactor as possible.

• Always wash hands after handling cultures.

• Wear a clean lab. coat or overall to protect cultures from microbes

on the skin, clothing, etc.

4. Ultrafiltration • Membrane Fouling, check for particulate matter adheres to surface

of membrane through the process

5. Vacuum dryer

• Do not use in an atmosphere containing corrosive gases, chemicals,

sea water, water, water steam, or where there is direct contact with
any of these.

• Do not use in a place subject to heavy vibration and/or shock.

• Do not use in an environment where flammable gas or explosive gas

exists. Usage may cause a fire or explosion. The products do not have
an explosion-proof construction.

• The product should not be exposed to prolonged sunlight. Use a

protective cover.
 • Remove any sources of excessive heat

6. Centrifugation

• Tightly seal all tubes and safety cups.

• Close lid during operation

• Allow to come to complete stop before opening.

• Only use manufacturer compatible centrifuge, rotor, buckets, caps,

and adapters.

9.3 Environmental impact.

Since our by product consist of acetic acid and are classified as hazardous by 2012
OSHA Hazard Communication Standard. Therefore , Hazard identification such as
(Flamable liquid and vapor) and (causes severe skin burns and eye damages) logos
will be labeled in the storage of the waste . Storage will be locked up and stored in a
corrosive area. A well ventilated place and ensure tightly closed at all times. Also will
be kept away from heat and sources of ignition. For Disposal , the contents will be
disposed to an approved waste disposal plant . In this case , we have chosen activated
sludge treatment. This is because , 99% of the acetic acid could be decomposed in 7
days under anaerobic conditions. This could prevent environmental impact .
 10.0 CONCLUSION

S. thermophilus has produced 2.408 g/L of high molecular weight HA 1000

kDa under optimal conditions in fed-batch fermentation. The purification method

developed have removed exothermic material, proteins, nucleic acids and other

impurities and hence high recovery (70 %) of HA was obtained. The recovery process

used is simple and economical by use of trichloroacetic acid (0.1%), charcoal

treatment, filtration and diafiltration. HA purified by this purification procedure

yielded a high purity (99 %) HA and this method could be useful for industrial

purification of clinical grade HA. It also shows that the cost of downstream

processing in the production of HA is significant because of the high cost of

consumables associated with the tangential-flow and polishing filtration steps. A

sterile, high MW, and highly pure product for medical applications show how a more

extensive purification section can be economically beneficial when the final product

can be sold at a substantially higher price.

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