Biorefinery-fermentation of Agro-wastes by Haloferax lucentensis GUBF-2 MG076878 to Haloextremozymes

for use as biofertilizer and biosynthesizer of AgNPs

Sanket K. Gaonkar and Irene J. Furtado*

Department of Microbiology, Goa University, Goa, India
*Corresponding Author: I. J. Furtado
[Email: ijfurtado@unigoa.ac.in]

Sanket K. Gaonkar

Research Scholar, Department of Microbiology

Goa University, Goa, India

[Email: micro.sanket@unigoa.ac.in]

Irene J. Furtado

Professor, Department of Microbiology

Goa University, Goa, India

[Email: ijfurtado@unigoa.ac.in]
ESM 1 a) Growth of Haloferax lucentensis GUBF-2 MG076878 in modified medium-5 with subculture 1, 2, 3, and
4. b) The CFFH from three subcultures showing constant protease activity on 20% NSM + 0.5% gelatin, c) The
CFFH from three subcultures showing constant lipase activity on 20% NSM + 0.5% Tween 80.

ESM 2 Concentration of crude haloextremozymes protease and lipase in CFCH and CFFH using tangential flow
filtration unit (a).The retentate and permeate of CFCH and CFFH in comparison with the crude extract (b). Gelatin
well assay showing the zone of protease activity (c, left upper and lower panel) and Tween 80 well assay with zone
of  lipase activity (c, right upper and lower panel) in 100μl each of retentate (R), Permeate (P) and Crude (C); B-
Blank. Silver stained 10% native gel with protein bands derived from CFFH  (d), Lane 1-Marker, Lane 2- R, Lane 3-
P, Lane 4-C) and CFCH (f), Lane 1- Marker, Lane 2- R, Lane 3 and 4- B, Lane 5-P, Lane 6-C ). In-gel protease
activity of rententate from CFFH using Coomassie stained method (e, Lane 1, 2 and 4- B, Lane 3- R) and lipase

activity (g) by gel overlay assay of the permeate from CFCH on Tween 80 agar showing the zone of precipitation.  

ESM 3 Effect of CFFH on Rice seed germination (Korgut)

Treatment Root length (cm) Shoot length (cm) Fresh weight (g) Dry weight (g)
Water 5.03 ± 0.20 5.02 ± 0.02 0.053 ± 0.002 0.0140 ± 0.0006
CFFH 6.72 ± 0.11 5.42 ± 0.25 0.074 ± 0.002 0.0160 ± 0.0005
NaCl (0.5%) 6.34 ± 0.10 4.96 ± 0.04 0.068 ± 0.003 0.0150 ± 0.0009
Each value is the mean ± SD of three individual experiments with 10 replicates (n=10).

ESM 4 Rice seed (Korgut) germination test.

ESM 5 Growth characteristics of rice plants (Korgut) by direct application of seeds under four different treatments.

Treatment Root length (cm) Shoot length (cm) Fresh weight (g) Dry weight (g)
1 5.64 ± 0.36 7.41 ± 0.37 0.83 ± 0.24 0.18 ± 0.61
2 6.49 ± 0.35 9.00 ± 0.72 0.94 ± 0.26 0.26 ± 0.52
3 7.50 ± 0.23 14.9 ± 0.96 0.98 ± 0.38 0.28 ± 0.92
4 7.36 ± 0.10 14.7 ± 0.93 0.97 ± 0.46 0.27 ± 0.84
1) 0.06 g each of potash and superphosphate, 2) 0.12 g cell-free feather residues, 0.06 g each of potash and superphosphate, 3) 0.12 g urea, 0.06 g each of potash and superphosphate and 4) 15 mL

CFFH, 0.06 g each of potash and superphosphate. Each value is the mean ± SD of three individual experiments.
ESM 6 Cell-free COC hydrolysate as biosynthesizer of AgNPs. 1mM AgNO3 (a) , AgNPs in solution (b), COC
hydrolysate (c), UV-vis scan: Before (d )and after (e) formation of AgNPs.