The document describes a biological process for producing riboflavin using recombinant Bacillus subtilis. Key points include: 1) B. subtilis converts glucose directly into riboflavin through fermentation, producing up to 16g/L of riboflavin crystals. 2) Downstream processing includes centrifugation to recover impure crystals, acid washing, and drum drying to produce feed-grade and pharmaceutical-grade riboflavin. 3) The biological process uses renewable resources and has lower environmental impact than chemical synthesis methods.

1. A Biological process for the
production of Riboflavin
Team Riboflavulous
Team 1 members
Priyesh Waghmare
Yixue Chen
Rebecca Milburn
Madhunika Padmanabha
Sharath Sathyan

2. Outline:
 Introduction
 Market
 Chemical Vs biological method
 Environmental impact
 Main process
 Waste treatment
 Mass balance
 Merits of our system
 Future prospects

3. Introduction to riboflavin (1)
 Riboflavin ( vitamin B2)
 Molecular formula C17H20N4O
 Yellow-orange
 Sparingly soluble in water
 Forms crystals in <30ºC water
 Light-sensitive

4. Introduction to riboflavin (2)
 Precursor of Co-enzymes
- flavin adenine dinucleotide (FAD)
- flavin mononucleotide
 Deficiency results in metabolic and skin disorders
 Riboflavin is mainly used as a food supplement for
both human and animals.

5. Market
 World demand is estimated to be at 6,000 tons p.a.
 Major producers are Roche, BASF and China’s
Hubei Guangji Pharmaceutical
 Feed-grade (80% ) sells at US $30/kg
 pharmaceutical-grade (98%) sells at US $50/kg.

6. Chemical v/s Biological Process
Chemical Process Biological Process
Glucose
K arabonate
Feed + Water
Ca arabonate +Innoculum
Ca ribonate
Ribonolactone
Ribose
Ribitylxylidine
Riboflavin
Phenylazo
Riboflavin

7. Riboflavin production (Biological process)
 Our design involves a single-step biological process
 Recombinant Bacillus subtilis is a gram positive,
aerobic bactiera that converts glucose directly into
riboflavin
 Recombinant B.subtilus can yield up to 16g/L
riboflavin in 48hours
 Riboflavin is sparingly soluble and forms crystals in
the fermentation broth
 Bacillus subtilis is much small than the produced
riboflavin particles, making downstream purification
easier

8. Life cycle assessment (LCA) of Riboflavin
Production
Type of process Chemical Biological
Raw materials (%) 100 150
Non-renewable raw 100 25
materials(%)
Energy(%) 100 90
Emissions of VOCs(%) 100 50
Emissions to water(%) 100 33
(Organisation for Economic Co-operation and Development., 2001)
 The biological method uses mainly renewable resources
 Less amounts of energy used in biological method
 Air and water are contaminated to a lesser degree by the biological process

9. Environmental Fingerprint

10. Overall Riboflavin Production Process

11. Feed-grade production

12. Pharma-grade production

13. Material Recycling

14. Energy Recycling

15. Waste Treatment – HCL gas
• HCL gas emitted in the process is removed
by scrubbers.
• In the packed tower, HCL gas flows
upward through a packed bed (provides
close gas-liquid contact) while the
scrubbing liquid flows downward by
gravity over the packing.
• The internal components of the tower
consist of a packing support plate, a
packed bed, a liquid distributor and a mist
eliminator.

16. Waste Treatment – Biomass waste
 Biodegradable organic waste is collected
and put into an anaerobic digestion
tank
 The material is broken down by
bacteria in the absence of oxygen
 Biogas product is cleaned, compressed
and sent to a CHP plant
 Solid digestate is also produced and can be used as
fertilizer

17. Used Charcoal- Thermal regeneration process
 Adsorbent drying at approximately 105 °C
 Desorption and decomposition at 500–900°C
under an inert atmosphere
 Residual organic gasification by carbon dioxide
at 800°C

18. Mass Balance
Spent Biomass 4.7
Feed (3.3) Nitrogen 22.6
Inoculums (5) Oxygen 5.2
Fermentation
Water 2.5 CO2 3.65
98.55
Differential
Air 30 Centrifugation
Water 84.5
Waste Water 7.35
All Measurements in tones Impure Riboflavin Crystals 2

19. Mass Balance
Impurities (0.4)
Riboflavin 1.8
Impure Riboflavin Crystals 2 Acid Wash
Dilute HCL 0.2
Dilute HCL 1.4 Acid + Water+
Trace amounts
Biomass 0.2
Feed Grade Drum Dryer
Riboflavin 1.6
1.6
All Measurements in tones

20. Differential Centrifugation:
Foodec decanter centrifuges focus on
 Hygiene
 Reliability
 Easy access
 User friendly
 Low noise level
Alfa Laval Foodec decanter
centrifuges - used for
pharmaceutical applications that
comply with strict sanitary
regulations.

21. Sizing & Cost:
Fodec 800
Capacity Depends on
application
G-force max 3243
Bowl material Duplex
stainless
steel
Other weight AISI 316
parts
Weight Kg 13000
(28860 lbs)
Installed Power kW 132-250
(140-
330Hp)
Sound Pressure dB(A) re.20lpa 89
Level
Cost 30,000 US
Dollars

22. Downstream Processing Equipments:
Candle filter System:
•Particulate removal system.
•Ceramic Filters
•Cyclones
•Residues up to 1ppm removed
Candle Filter System:
Stirred Tank Fermenter:
•Coiled tube for Heating & cooling
•Uniform mixing
•Better fermentation
•Defoamer
Stirred Tank Fermenter:

23. Merits of our system
 A simple one-step biological process
 Bacillus subtilis requires a relatively unrefined
growth conditions
 No harmful chemicals used in process
 >90% culture medium recycle
 >90% hydrochloric acid recycle
 Used biomass recycling as nutrients
 Energy recycling system in place
 Large storage tank in place to make full usage of
downstream process equipment

24. Future Prospects
 To genetically modify bacillus subtilis to create a
strain that has a higher product yield (>16g/L)
 To replace part of the glucose feed with cheaper
organic residues like rotten potatoes/oranges
 To develop a process for purification of pharma-
grade riboflavin with fewer steps