stream 

 

.. 

 

Furfural 

 

Biological production of a hydrocarbon fuel intermediate polyhydroxybutyrate (PHB) 

from a process relevant lignocellulosic derived sugar 

Wei Wang, Ashutosh Mittal, Ali Mohagheghi, David K. Johnson 

National Renewable Energy Laboratory, Golden, CO 80401
 

Abstract Growth of A. eutrophus in pretreated 

saccharified lignocellulosic slurry* 

Sugar utilization 

30.0 2.0

PHAs are synthesized by many microorganisms to B 25.0 serve as intracellular carbon storage molecules. In 3 

1.5 

15.0 1.0 

10.0 

0.5 

5.0 

0.0 0.0 

0 20 40 60 80 100 120 

LB 

Mock slurry+LB 

Slurry+LB 

Acetate concentration (g/L)

some bacterial strains, PHB can account for up to 2.5 

Glucose, Xylose

concentration (g/L)

20.0 

80% of cell mass. In addition to its application in the 2 packaging sector, PHB also has great potential as OD600nm

an intermediate in the production of hydrocarbon 

fuels. PHB can be thermally depolymerized and 

1.5 

1 

decarboxylated to propene which can be upgraded 0.5 to hydrocarbon fuels via commercial 

oligomerization technologies. Alcaligenes 

eutrophus is the microorganism that has been most 

extensively studied and used for PHB production on 

an industrial scale; However the substrates used for 

producing PHB are mainly fructose, glucose, 

Time (h) 0 

Mock sugar Glu Untreated Glu AC treated Glu 0 20 40 60 80 100 120 Mock sugar Xyl Untreated Xyl AC treated Xyl 

Time (h) Mock sugar Ace Untreated Ace AC treated Ace 

* Hydrolysate slurry was saccharified with Ctec2 Htec2 enzyme from Novozyme 

Better cell growth was found in carbon treated slurry 

A. eutrophus did not grow in the original however glucose utilization was much slower than

saccharified slurry or even in the 2-fold diluted 

slurry. This low viability of cells suggests that the 

strain is not tolerant to the high concentration of 

toxic compounds in the hydrolysate slurry 

sucrose, fatty acids, glycerol, etc., which are 

expensive. In this study, the strain was grown on 

pretreated lignocellulose hydrolyzate slurry and 

evaluated in terms of cell growth, sugar utilization 

and PHB accumulation. In addition, the mechanism 

of inhibition in the toxic hydrolysate generated by 

sugar control indicating other inhibition still existed. 

Growth in slurry saccharified 

the pretreatment and saccharification process of with advanced enzyme

biomass, was also investigated. Treatment of acid hydrolysate Cell growth Sugar u=liza=on	
   slurry with activated carbon 3.00	
  

LC-MS analysis Sugar to hydrocarbon fuel 2.50	
   OD600 2.00	
  

0

5

10

15

20

25

30

Glucose	
  conc	
  (g/L)

process flow diagram 1.50	
  

1 

2 

3

4 

5 

6 7 8 9 

10 

11 1213 1415 

16 

17 

18 19 20 21 22 23 24 25 

26 

27 

28 

29 30 

31 

32 

33 

34 

35 

36 

0 10 20 30 40 50 Time [min] 

1 

2 34 5 6 7 8 9 

10 

11 1213 1415 16 17 18 19 20 21 22 23 24 25 

262728 29 30 313233 3435 

36 

Intens. 

mAU 

Furfural 

Levulinic HMF

acid Vanillic 

acid 

Hydroxylbenzoate 

p- coumaric 

Ferulic 

acid 

Syringaldehyde 

Acetic 

acid 

Intens. 

mAU 

2000 

1.00	
  

1500 0.50	
  

0.00	
   1000 0 20 40 60 80 100 120 0 24 48 72 9 Mock sugar Time	
  (h) Mock Suga Time	
  (h) Untreated Ctec2 Htec2 saccharified	
   500 Carbon treated Carbon treated Ctec2 Htec2 saccharified	
   Ctec3 Htec3 saccharified Ctec3 Htec3 sacchrified	
   carbon treated Ctec3 Htec3 saccharified carbon treated Ctec3 Htec3 saccharifie 0 

* Hydrolysate slurry (4x diluted) was saccharified with Ctec3 Htec3 

1250 enzyme from Novozyme Fermentation 

1000 

A.eutrophus grew well in new enzyme saccharified 

slurry (4x diluted). Similar cell growth and sugar 

utilization rate, and almost same PHB content of 

33% as in mock sugar medium were achieved. 

Conclusions 

750 

500 

250 

0 

0 10 20 30 40 50 Time [min] 

(top) before treatment (bottom) after treatment 

Materials and methods LC-MS results showed that most of the inhibitors in

the slurry including furfural, HMF, vanillic acid, coumaric 

Microorganisms acid etc. have been removed by carbon treatment

Alcaligenes eutrophus NCIMB 11599 

Cell growth in carbon treated PHB analysis 

The PHB content of bacterial cells was determined by a hydrolysate slurry (4x) 

quantitative method that uses HPLC to measure the CA A 

2.5

formed by acid-catalyzed depolymerization of PHB. Mock sugar 

Mock sugar+LB Untreated Carbon treated

Untreated slurry

AC treated slurr

+LB 

y+LB 

2

Briefly, freeze dried bacterial cells (15 to 50 mg) were 

digested in 96% H2SO4 (1 mL) at 90°C for 1 hr. The 1.5 OD600nm

reaction vials were then cooled on ice, after which, ice­ 1 

cold 0.01N H2SO4 (4 mL) was added followed by rapid 0.5 v Lignocellulosic hydrolysate can be used as mixing. The samples were further diluted 20- to 150-fold 0 substrate for biological production of PHB. 0 20 40 60 80 100 120 

Time (h) 

with 0.01N H2SO4 and analyzed by HPLC. 

v Carbon treatment was effective in removing most 

chemical inhibitors in the acid hydrolysate slurry. 

Although these chemicals proved to be inhibitory 

to A. eutrophus they were not the vital factor 

leading to inhibition for the microorganism; 

v The good performance of strain in slurry 

saccharified with advanced enzyme indicated 

some compounds generated in saccharification 

process in first slurry, most likely oligosaccharides, accounted for much of the inhibition; 

The information contained in this poster is subject to a government license. 

The 36th Symposium on Biotechnology for Fuels and Chemicals, Clearwater This work was supported by the U.S. Department of Energy with 

the National Renewable Energy Laboratory NREL/PO-2700-60861 beach, FL, April 28 - May 2, 2014