Environmentally Relevant Technology Based

on Protein-Enhanced Surfactants

Michael Goldfeld, Carl Podella, Jack Baldridge

&Chris Rulison

Augustine Scientific, Newbury, OH

, Irvine, CA, www.abiocat.com

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ABC’s PROFILE

Advanced BioCatalytics Corporation - Develops, manufactures & markets non-toxic & environmentally friendly products for cleaning & remediation- Strategic Manufacturing Agreements with Major Food Companies

Major applications: - industrial cleaning & degreasing;

- wastewater treatment;- soil & water bioremediation;

- oil spill clean;- odor control

reaching out to EOR, agriculture, animal care, skin care, etc.

R&D conducted in-house & through collaboration with UC & contracts with certified/licensed labs. Pilot & field tests on consumer sites.

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CORE TECHNOLOGY :

synergy betweensurfactants

& proteins

4

Traditional approach:

surfactants affect proteins,

e.g. unfolding, inhibiting enzymes.

ABC’s approach: proteins empower

surfactants

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A technologyborne on the premise

that we should first,

DO NO HARM …

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ABC’s proteins are baker’s yeast

stress exo-proteins

Heat-Shock Proteins, HSP

small proteins (5-30 kDa) released by living yeast

in response to a mild heat shock

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SURFACTANTS:Synthetic & Bio-derived :

anionic, cationic, non-ionic & amphotericform

PROTEIN-SURFACTANT COMPLEXES

HSP + S → PSC

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PSC-s:- Are stable & functional from pH 1 to 13

- Retain surface activity after heating at 90ºC for96 hours

- Compatible with strong oxidants (such as H2O2, bleach, I2 …)

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The brand name of the core product is

Accell®& there is a suite of related formulations:

Accell-3®, Accell Clean®,

Accell Clean® Marine, Accell Clean® Plus, Accell® Green, Accell Clean® DWD, Accell Clean® SWA,

Accell Clean® DPP …

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Accell®

- is a homogeneous, amber colored, water borne liquid, buffered at pH~5- NO VOC, bacteria or living cells, little amylase & no other enzymatic activity, & is manufactured on food processing plants.

- non-toxic to animal & fish, marine life, minimal to non-irritant, environmentally safe, & bio-degradable- only ingredients recognized as safe in food grade applications according to FDA 21 CFR 178.3400 and/or found on the GRAS list.

- No need for protecting attire with Accell®

Approvals,Certifications & Listings:NSF: Accell® & Accell® -3: Standard-60 Certification (2007 & 2011) for

treatment of potable water. IMO: Accell Clean® Marine & Marine Plus certified for chemical cargo tank

cleaning (2009 & 2011) ,

US Coast Guard approval for Accell Clean ®

Deepwater Horizon MC252 Unified Command approved Accell Clean ® for oil spill decon.

Accell Clean® SWA & Accell Clean® DWD listed on the EPA-NCP listing for surface washing and as oil dispersant.

Accell Clean® G received the Green Seal certification (2012).

California Dept. of Fish & Wild Life licensed Accell Clean® SWA for oil spill cleanup

Accell® 3 IBAMA Registration – Brazil

Oman Ministry of Environment & Climate Affairs approval for Accell Clean ®

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Formation of PSC-s achieves two goals: Empower synthetic surfactants: reduces ST, IFT, CMC,

facilitates wetting, spreading, penetration solubilization, resulting in more efficient cleaningThis is an immediate, short-term effect

PSC-s activate bio-processing of contaminants by the existing aerobic microflora , as

in WWT, or oil spilled in ocean, or as a follow-up effect after regular cleaning.

This is a longer-term effect associated with microbial cycle

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BENCH METHODS:Interfacial (IFT) & surface tension (ST);

& Contact angle measurementsPendant Drop:Oil-water IFT &

drop volume

CCD Camera

Pendant drop IFT & drop volume analysis

Capillary5L oil droplet

Wetting & uptake

CCD CameraDrop volume & contact angle

analysis

Contact angle & drop volume:

Spread only, no uptake: volume constant

Spread & uptake: volume reduced

Green leaf or skin

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0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

1.0 10.0 100.0 1000.0 10000.0

IFT, mN/m

LAS concentration, ppm

IFT as function of BioSoft D-40 (LAS) concentration:

LAS alonebaseline

LAS-CF 1:1

LAS: sodium dodecyl benzene sulfonate

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0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

3 30 300

IFT mN/m

Sodium Laureth Sulfate, ppm

Aqueous Steol CS -270 against Canola Oil Interfacial Tension vs. Concentration Data

Steol CS-270 Alone

Steol CS-270 in 233 ppm CCF-2013

Steol CS-270 in 1200 ppm CF-2013

0.52

3.83

CMC,  ppm

157.5

29.7

27.6

Protein:CF – Clear Ferment

CCF – 5.15×membrane-

concentrated CFConcentration of CCF=233 ppm taken 1/5.15 of the concentration

of CF = 1200 ppm

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PROTEIN SYNERGIST REDUCES IFT & CMC

With some surfactants,

CMC is reduced by 1-2 logs, & lower pre-CMC IFT

7.72146.11:2 LO -CS230

333Steol CS-230(Na laurethsulfate)

99.78Ammonyx LO(lauramine oxide)

+proteinno proteinSurfactant

CMC, ppm

13.031240

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Small Protein Effect on the Interfacial Tension

Sodium Lauryl Ether Sulfate

± Lysozyme (LZ)

Shift to lower CMC

Lower IFT in pre-CMC area

LZ}

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Accell Clean®

cleaning power in removal of Dieselcontaminant from bilge water, as compared to a common highly alkaline cleaner

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 500 1000 1500 2000 2500 3000

Time, min

BILGE CLEANINGPendant Drop Method

CommercialCleanerpH 13

Accell® CleanpH 8

Die

sel D

rop

Volu

me,

L

Accell:

95% cleaning in 20 minat neutral pH

Commercial cleaner: 70% cleaning after 50 hrs at pH 13

Oil-water IFT & drop volume

CCD Camera

Pendant drop IFT & drop volume analysis

Capillary

5L oil droplet

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0.350

0.400

0.450

0.500

0.550

0.600

10 15 20 25 30 35 40 45

IFT,

mN

/m

% CF

Tuning IFT with CFAccell® Clean SWA:

20% CF

Accell® Clean SWA

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Enhancing Wettingto facilitate

Spread & Foliar UptakeContact angle & drop volume:

Wetting & uptake

Drop volume & contact angle

analysisCCD Camera

Spread only, no uptake: volume constant

Spread & uptake: volume reduced

GREEN LEAF

0.25% NPE droplet on Cabbage Leaf 0.25% ABC droplet on Cabbage Leaf

COMMERCIAL DETERGENT vs. ABC PRODUCT

Comparison of Wetting & Uptake for Two Aqueous Solutions on Tomato & Cabbage Leaves

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0.70

0.80

0.90

1.00

0.0 100.0 200.0 300.0 400.0 500.0 600.0

Dro

p Vo

lum

e

L

Time, sec

Drop Volume Kinetics After Application to a Cabbage Leaf

Wetting & Uptake on Plant Leaves: Effect of Protein Synergist

Surfactant

Surfactant + Protein Synergist

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y = -0.0002x3 + 0.021x2 - 1.758x + 129.14R² = 0.9982

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50

60

70

80

90

100

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120

130

0 10 20 30 40 50 60 70 80

sec

Clear Ferment 5x Content, %

Time of Total Uptake (>95%)

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GRAPE LEAF UPTAKE OF ZINC MICRONUTRIENT: EFFECT OF ABC’s SwiftWet™

Major CA agribusiness co conducted tests on vine grape. Zn is a micronutrient routinely sprayed on leaves using Foli-Gro Zinc 7% with ABC’s SwiftWet™ or other adjuvants. Zn was applied at a consistent rate in every treatment, while SwiftWet™ was applied at different concentrations.

SwiftWet™ was plant safe (no observed phytotoxicity) in every treatment.

6-fold Zn uptake in dose-rate dependent manner vs. Control (no adjuvant).2-fold increase with other adjuvants

SwiftWet™

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Skin Care Implications

Dro

p Vo

lum

e,

L

Time (seconds)

Drop Volume Kinetics on Human Skin80:1 Dilution of 7-ABC-26-11-SK-1

80:1 Dilution of 7-ABC-26-11-SK-2

80:1 Dilution of 7-ABC-26-11-SK-3

80:1 Dilution of 7-ABC-26-11-SK-4

80:1 Dilution of 7-ABC-26-11-SK-5

80:1 Dilution of 7-ABC-26-11-SK-6

80:1 Dilution of 7-ABC-26-11-SK-7

80:1 Dilution of 7-ABC-26-11-SK-8

1: CS-230 Na laureth sulfate

2: CS-230+StepanMild L3, Lauryl lactyl lactate

3: (1) +CF4: (2) + CF

7: (5) + CF

8: (6)+CF

6: (5) + StepMild L3

5: StepanMild SL3-BA, Di-Na laureth sulfosuccinate

(degrees)

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Compatibility with strong oxidants (bleach, HP, I2, Peracetate) allowed for formulation of

disinfectants combining H2O2 & PSC

Plate Count, 10-6 CFU/mL

Microbe Agent Time Zero 1 min 2 min 5 min 10 min

S. aureus

Control3% H2O2

10.4 12.0 8.80 3.86

4% PSC3% H2O2

19.9 0.109 0.0113 0.00

E. coli

Control3% H2O2

19.9 17.1 6.80 1.36

4% PSC3% H2O2

17.3 0.68 0.04 0.00

ProDipTM Teat Dip for Dairy FarmingContains HP & human skin care grade surfactants and is a step forward towards human skin care products

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Root Promotion in SpinachPSC-s produced ca. 3x the root mass compared to the control after 35 days of treatment at 25 ppm Accell®3

CHEMICAL TANKERS FLEETSCleaning of chemical cargo tanks in ocean fleets must meet highest standards. VOC solvents are prohibited & alkaline cleaners damage zinc coatings.

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CHEMICAL TANKERS FLEETS Accell Clean® Marine, IMO certified, saves time, needs fewer & shorter wash cycles to clean oils, reduces the waiting time outside harbor while washing tanks to meet inspection standards, & is cost effective. 

23,6601,500Grand Total Cost per Tank, $22,000n/aDaily Loss of Earnings, $1,6601,500Total Chemical Cost $

5631Total (hrs)

Other Chemicals(pH 12-13)

AccellClean® M

pH 8

* Cleaning according to Methanol Standard after Plant or Petroleum oil in Zinc or Epoxy Coated 3000-m3 Tank

Comparative costs: Accell Clean® Marinevs. other chemicals*

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AccellClean

Accell® Clean Marine• Superior tank

cleaning:

• Superior safety for ship’s cleaning crew

• Superior safety for the environment

Safely & efficiently cleans zinc, epoxy coated & stainless steel tanks

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CLEANING OF INDUSTRIAL FOOD PROCESSORS:ROTISSERIE OVENS

Due to the stability of PSC to certain oxidants, ABC developed products with H2O2 designed for cleaning of tenacious soils from stainless steel surfaces in  industrial rotisserie ovens:

After ABC’s product sprayed scrubbed & wiped.

After processing ca. 200 chickens. Surfaces are heavily stained with partially caramelized & carbonized material.

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ABC’s product not only cleans, but also reduces adhesion & prevents caramelization & carbonization of organic waste on the surface:

In subsequent cycles, lesser accumulation of tenacious carbonized stuff, hence less labor on cleaning

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Confronting Oil Spills

• Herding & Collecting

• Dispersing

• Cleaning soil, beaches, deoiling tar sand

• Cleaning Ships & Equipment

• Bioremediation

Accell Clean® SWA - Herding Oil on Waters – 1st response to a spillhelping to collect oil

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Heavy Ventura Crude

Luisiana Light Crude

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Accell Clean® DWD has been included in the EPA NCP list for petroleum oil DISPERSANTS, & Accell Clean® SWA – in the list of the agents permitted for cleaning of equipment, boats, soil and other solid surfaces.

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Comparison of Small-Scale Dispersant Testing Methods to Ohmsett:Effect of Dispersant Type and Oil Properties

for: Bureau of Safety and Environmental EnforcementU.S. Department of the Interior

by: S.L. Ross Environmental Research Ltd.Ottawa, ONJune 2013

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With a Highly Caustic Cleaner 6 men, 2 hours, wiping

With Accell® Clean: 2 men, ½ hr, no impingement

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Oil Contaminated Beach CleaningUsing Accell Clean

Same approach is valid to processing tar sands for oil recovery

Extractable oil:

in non-cleaned sand (black): (2.1±0.5)%in Accell-cleaned sand: < 0.1%

Result: Below detect in 3min 14sec

All operations occur at ambient temperature. 90% of water is recycled. 10% is released in clean wet sand.Separated oil is removed & disposed, or may be collected & recycled

Beach decontamination by Proven Technologies, Inc., (Houston, TX) Beach Cleaner machine

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Aver

age

barr

els

per D

ay

Month

TEXAS - Pecan Gap - Well D7Pre//Post Treatment Production Treatment Date Sept 12th, 2013

1 week Treatment

Texas Oil Field, 2900 ft Well Production Before and After Treatment with ABC’s Product

PARAFFINS CLOG OIL WELL, HARM PRODUCTION. ABC’s AGENT UNSEALS PRODUCTION WELL

5% Accell Clean DPP 3x pipe volumeCirculated for 1 week

IN PROGRESS

5×production

boost

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PSC-s display two effects: Empower synthetic surfactants: reduce ST, IFT, CMC, facilitate wetting, spreading,

penetration, solubilization, uptake, etc.This is an immediate, short-term effect.

Activate bio-processing of contaminants by aerobic microflora, in WWT, or petroleum oil spilled in the ocean, or as a follow-up effect after regular cleaning.

This is a longer-term effect associated with microbial cycle.

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TREATMENT OF INDUSTRIAL EFFLUENTS & WASTEWATER

involves the effect of PSC on the core bacterial metabolism

Kikkoman Foods-WI, for 10+yrs, applied Accell® to pre-treat its effluentAccell® application reduced BOD, TSS & costs of aeration, & eliminated malodor. The company complied with the regulations & even could sell some of their quota to other local industries, with no capital investments. 

Reduction in Sludge Production 30% to 50%

Reduction in Aeration Power Costs 25% to 50%

Increased Aeration Tank throughput 20% to 40%

Reduced Effluent Ammonia 30% to 75%

Reduced Effluent Phosphorus 45% to 65%

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WHAT IS THE REASON?

After the treatment began:An unexpected BOD reduction with a concomitant TSS reduction was observed. The increased microbial activity required to digest this level of BOD would normally result in a significant increase in TSS.

With Accell, a shift in the processing features observed:

In 3 yrs before the treatment: the rise & fall of BOD ran COUNTER to the rise & fall of TSS, as expected: with healthy microflora (summer), more nutrients assimilated, more biomass as TSS. A less active microbial system (winter), consumes less nutrient resulting in higher BODlevels, & less TSS.

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BIOLOGICAL EFFECT:Accell® as an Uncoupler of Bacterial Oxidation

MAINTENANCE

BIOSYNTHESIS:ANABOLISM

Endergonic rxns

ATP-ADP CYCLE

ATP

Exergonic rnsNUTRIENT OXIDATION:

CATABOLISM

Carbon O2

ADP + Pi

CO2

Exergonic rxns

uncoupler

Cell Interior Matrix

Outer Space

H+UNCOUPLER

ETC

ETC

Cell respiration: ATP-synthase, proton pump

& uncouplers

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H+ H+ H+

H+ H+

H+

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2-chamber set for studies of H+

transfer across a hydrophobic liquid polymer-supported membrane.

Nutrient bio-oxidation with Accell® is uncoupled from biomass accumulation, similar to standard dinitrophenol (DNP ) uncoupler, known to shuttle

H+ across membrane, thus dissipating pH & preventing energy supply as ATP.

Model membranes tests confirm the proton-leak effect of Accell®, similar to DNP

Conclusion: Accell® uncouples bio-oxidation from biomass growth.It does so by increasing membrane permeability for H+, thus suppressing pH & ATP formationLifting phosphorylation control speeds up bio-oxidation

H+ leak from acidic to alkaline cell is recorded as pH shift.

Regular functioning: cell is coupled

Nutrients are oxidized to CO2, while H+ pumped out of cell. pH is formed: more protons outside.

pH drives H+ flow to the cell interior through the channel formed by ATP synthase mushroom. Transfer of through this channel results in ATP formation.

H+ UNCOUPLER

Uncoupled Cells:

Uncoupler provides a shunt for H+ leak, no H+ flow through ATP-synthase, no ATP formed

Cells stop proliferating.Biomass growth inhibited.

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WASTEWATER LAGOONClark County, NV Sanitation District

Prior to treatment Post-treatment

6 months at 6 ppm Accell®

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SYNERGISTIC MICROBE-DETERGENT ENHANCEMENT OF SURFACTANCY:

Grease as a Source of Surfactant IFT drop & Accell® cleaning power are enhanced over the time in presence of activated sludge, as compared to sterile conditions.

Glucopon 425 N from Cognis, OH, oligomeric C10-16 alkyl D-glucopyranoside

Effect of yeast proteins & grease exposure on IFT (mN/m) in aqueous surfactant/bacon grease system:

Petroleum Oil Contaminated Soil & Sludge Bio-remediation

1Gal/8yd3wk

Bench Study: 7% Crude oil in soilAccell® 1/16 dilution

Regular watering to 11% moisture

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2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

-25

-20

-15

-10

-5

0

TPH degraded

% to regular watering

Canadian Soil Bioremediation Bench Test

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% TPH in soil (Type N, pretreated)

after 20 days remediation. Control: samples stored in

freezer

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Petroleum Oil 421 Bioremediation in Two Samples of Water Collected in the Port Fourchon Area, 21 Days Incubation at 24oC

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PINHEIROS RIVER PROJECTSão Paulo BRAZIL

REPORT ON THE TEST COMPLETED USING ACCELL® 3 FOR TREATMENT OF PINHEIROS WATER CANAL

Engeform 19/03/2013

6 Test Channels, 30 m long each

Downtown PinheirosShores

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PINHEIROS RIVER PROJECT

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5

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15

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25

30

35

40

45

50

INFLOW 15M OUTFLOW

TSS, mg/L45.2

14.311.75

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PINHEIROS RIVER PROJECT

0

0.5

1

1.5

2

2.5

3

INFLOW 15M OUTFLOW

Sulfides, mg/L

2.58

0.1 0.1

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● Complexes formed with surfactants by the yeast stress exo-proteins (PSC) display enhanced surface activity & more subtle effects on the microbial metabolism.

● PSC accelerate aerobic processing of organic contaminants in WWT, with a concomitant reduction in biomass growth (solid sludge) due to an uncoupling of bio-oxidation from endergonic processes.

● PSC stimulate biochemistries relevant to environmental technologies, such as bioremediation of petroleum oil contaminated water & soil.

● Other bio-implications include odor control, biofilm prevention, sewer line degreasing, & activation of interfacial enzymes, soil & animal treatment …

CONCLUSIONS

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Outside collaborators:Dr. Stuart Krassner, UC-Irvine; Jim Bethke, UC-RiversideDr. Chris Rulison, Augustine Sci., OH; Cpt. Raaamu Naidu Sanasi, MalaysiaDr. Tilak Nagodawithana, WI

Carl Podella, Founding VP, R & D

Bill Torres, President

Jack Baldridge, Founder, Chairman & CEO

Andy Michalow, Director - Business Development & New Technologies

Dr. Michael Goldfeld, Director - Corp. Research

Advanced BioCatalytics Team: