1.

5 PROCESS DESCRIPTION

In this plant, Aureobasidium pullulans ZX10 (Zou et. al. 2013) is used to ferment
xylose and glucose under batch and aerobic conditions, to yield polymalic acid with
the release of acetic acid, succinic acid, carbon dioxide and water as side products.

1.5.1 Pre-treatment of raw materials

Zou et. al. (2016) utilized corncob hydrolysates for polymalic acid production, but in
this plant rice straw was used as lignocellulosic feedstock for economic reasons and
since the sugar contents for both are comparable; 34.4% glucan, 19.7% xylan and
19.8% lignin (Uppugundla et. al. 2014). Rice straw is pre-treated using Ammonia
Fiber Expansion (AFEX) conditions as implied by Harun et. al. (2013) and the
resulting biomass is completely recovered since there is no wash stream. It is then
added into a bioreactor containing lignocellulotic enzyme system medium, where
glucose and xylose in the form of rice straw hydrolysates (RHS) are obtained. The
RSH are expected to contain mixed sugars with 72.1% glucose and 27.9% xylose
(Uppugundla et. al. 2014).

1.5.2 A. pullulans ZX10 Inoculation and Seed Fermenter

A. pullulans ZX10, the highest-PMA-producing-strain in the study done by Zou et. al.
(2013) was isolated from NRRL Y2311-1, from NRRL Culture Collection (Peoria,
IL). Stock culture was maintained on potato dextrose agar (PDA), on a PDA medium,
and stored at 4C. Seed culture was then inoculated into a seed fermenter containing:
glucose (20g/L), (NH4)NO3 (60g/L), KH2PO4 (2g/L), MgSO4 (0.1g/L), ZnSO4(0.1
g/L), KCl (0.1g/L) and CaCO 3(0.1g/L) which was used to maintain pH at 6 during
fermentation. The seed fermenter was left in aerobic conditions for 48 hours at 25C to
cultivate biomass.

1.5.3 Fermentation of RSH

The fermentation was carried out in a bioreactor in batch mode at 25C, pH 6 and
aerobic. The fermentation medium was prepared as follows: RSH (90 g/L), (NH 4)NO3
(60g/L), KH2PO4 (2g/L), MgSO4 (0.1g/L), ZnSO4(0.1 g/L), KCl (0.1g/L) and
CaCO3(0.1g/L). After 72 hours, polymalic acid (PMA), acetic acid, succinic acid,
biomass, water and carbon dioxide were released.
1.5.4 Separation and Purification of Polymalic Acid

The downstream processing of PMA fermentation to obtain final PMA product

includes cell separation by centrifugation, microfiltration for concentrating PMA
solution, alcohol precipitation for final PMA recovery and purification and spray
drying which releases PMA powder. The fermentation broth is first subjected to
centrifugation at 8000 rpm for 45 minutes to separate the biomass, followed by
microfiltration (0.45m membrane pore size). It is followed by ethanol precipitation
where anhydrous ethanol was added to the filtrate and incubated overnight at 4C. The
resulting precipitates and the mixture of ethanol-water is separated via decantation.
Ethanol was recovered and recycled via distillation. The PMA precipitates were
washed with water and spray-dried to form PMA powder, ready for shipping.
 REFERENCE

Zou, X., Zhou, Y. & Y, S.T. 2013. Production of Polymalic Acid and Malic Acid by
Aureobasidium pullulans Fermentation and Acid Hydrolysis. Biotechnology and
Bioengineering: 110(8): 2105-2113.

Zou, X., Yang, J., Tian, X., Guo, M., Li, Z. & Li, Y.Z. 2016. Production of polymalic
acid and malic acid from xylose and corncob hydrolysate by a novel Aureobasidium
pullulans YJ 611 strain. Process Biochemistry: 51: 16-23.

Harun, S., Balan, V., Takriff, M. S., Hassan, O., Jahim, J.M. & Dale, B.E. 2013.
Performance of AFEX pretreated rice straw as source of fermentable sugars: the
influence of particle size. Biotechnology for Biofuels: 6:40.

Uppugundla, N., da Costa Sousa, L., Chundawat, S.P., Yu, X., Simmons, B., Singh, S.,
Gao, X., Kumar, R., Wyman, C.E., Dale, B.E. & Balan, V. 2014. A comparative study
of ethanol production using dilute acid, ionic liquid and AFEX pretreated corn
stover. Biotechnology for Biofuels: 7:72.