A Biological process for the production of

Riboflavin

Team Riboflavulous

Team 1 members

Priyesh WaghmareYixue Chen

Rebecca MilburnMadhunika Padmanabha

Sharath Sathyan

Outline:

IntroductionMarketChemical Vs biological methodEnvironmental impactMain processWaste treatmentMass balanceMerits of our systemFuture prospects

Introduction to riboflavin (1)

Riboflavin ( vitamin B2)Molecular formula

C17H20N4OYellow-orange Sparingly soluble in

waterForms crystals in <30ºC

waterLight-sensitive

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.

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.

Chemical v/s Biological Process

Glucose

K arabonate

Ca arabonate

Ca ribonate

Ribonolactone

Ribose

Ribitylxylidine

Phenylazo

Riboflavin

Feed + Water

+Innoculum

Riboflavin

Chemical Process Biological Process

Riboflavin production (Biological process)

Our design involves a single-step biological processRecombinant 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

Life cycle assessment (LCA) of Riboflavin Production

Type of process Chemical Biological

Raw materials (%) 100 150

Non-renewable raw materials(%)

100 25

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

Environmental Fingerprint

Overall Riboflavin Production Process

Feed-grade production

Pharma-grade production

Material Recycling

Energy Recycling

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.

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

Used Charcoal- Thermal regeneration process

Adsorbent drying at approximately 105 °CDesorption and decomposition at 500–

900°C under an inert atmosphereResidual organic gasification by carbon

dioxide at 800°C

Mass Balance

Fermentation

Feed (3.3)

Inoculums (5)

Water 2.5

Air 30

Nitrogen 22.6

Oxygen 5.2CO2 3.65

Waste Water 7.35

98.55

Differential Centrifugation

Spent Biomass 4.7

Water 84.5

Impure Riboflavin Crystals 2All Measurements in tones

Mass Balance

Impure Riboflavin Crystals 2 Acid Wash

Impurities (0.4)

Dilute HCL 1.4

Dilute HCL 0.2

Drum Dryer

Riboflavin 1.8

Acid + Water+ Trace amounts Biomass 0.2

Feed Grade

Riboflavin 1.61.6

All Measurements in tones

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.

Sizing & Cost:

Fodec 800

Capacity Depends on application

G-force max 3243

Bowl material Duplex stainless steel

Other weight parts

AISI 316

Weight Kg 13000 (28860 lbs)

Installed Power kW 132-250 (140-330Hp)

Sound Pressure Level

dB(A) re.20lpa 89

Cost 30,000 US Dollars

Downstream Processing Equipments:

Candle filter System:•Particulate removal system.•Ceramic Filters•Cyclones•Residues up to 1ppm removed

Stirred Tank Fermenter:•Coiled tube for Heating & cooling•Uniform mixing•Better fermentation•Defoamer

Candle Filter System:

Stirred Tank Fermenter:

Merits of our system

A simple one-step biological processBacillus subtilis requires a relatively

unrefined growth conditionsNo harmful chemicals used in process>90% culture medium recycle>90% hydrochloric acid recycleUsed biomass recycling as nutrientsEnergy recycling system in placeLarge storage tank in place to make full

usage of downstream process equipment

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