Supplementary Data
Changes in the physicochemical properties and enzymatic activity of waste during
bioreduction of pig carcasses
Ceri L. Gwythera*, David L. Jonesa, Christoph Gertlerb, †Gareth Edwards-Jonesa, A.
Prysor Williamsa
a
School of Environment, Natural Resources & Geography, College of Natural Sciences,
Bangor
University,
Gwynedd,
LL57
2UW,
UK;
c.l.gwyther@bangor.ac.uk.
d.jones@bangor.ac.uk, prysor.williams@bangor.ac.uk
b
School of Biological Sciences, College of Natural Sciences, Bangor University,
Gwynedd, LL57 2UW, UK; c.gertler@bangor.ac.uk
*
Corresponding author: Tel.: +44 1248 383062; Fax: +44 1248 354997; E-mail:
c.l.gwyther@bangor.ac.uk
Table S1: DNA extraction method for bioreduction microbial communities; based on
Anderson and McKay (1983). All centrifugations were done at 4 °C in an Eppendorf
Centrifuge S810R (Eppendorf UK Limited, UK).
Step
Volume
Centrifuge 1 ml defrosted sample
13,000 g, 10 min
Add STE-buffer (0.1 M NaCl, 10 mM Tris-HCl pH 8.0 and 1 mM
EDTA pH 8.0)
700 µl
Add lysozyme (10 mg ml-1 in 25 mM Tris, pH 8)
18.4 µl
Vortex for 2 s and incubate
37 °C, 30 min
Add Tris-EDTA (10mM Tris-HCl, and 1 mM EDTA, pH 8.0)
73.5 µl
Add sodium dodecyl sulphate (SDS) (20% [wt/vol] in 50 mM Tris-20
mM EDTA, pH 8.0)
44 µl
Vortex for 2 s and invert reaction tubes 6-8 times
Incubate
50 °C, 60 min
Centrifuge
10,000 g, 10 min
Transfer supernatant into a new 1.5 ml reaction tube
420 µl
Add 5 M NaCl
42 µl
Add phenol-chloroform-isoamylalcohol (25:24:1)
420 µl
Invert reaction tubes 15-20 times
Centrifuge
10,000 g, 10 min
Transfer upper aqueous phase into new reaction tube
200-350 µl
Add chloroform-isoamylalcohol (24:1)
1 volume
Centrifuge
10,000 g, 10 min
Transfer upper aqueous layer into new reaction tube
100-200 µl
Add isopropanol
1 volume
Incubate
4 °C, 12 h
Centrifuge
20,000 g, 20 min
Carefully remove the supernatant and keep the pellet
Open reaction tubes, cover with a sheet of paper and air-dry in a fume
hood
1h
Remove remaining excess isopropanol using a DNA concentrator
(Savant DNA120 Speedvac, Thermo Scientific, UK)
20 min
Resuspend pellets in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0)
30 µl
Figure S1: The change in microbial communities over time in two bioreduction vessels containing pig carcasses. T = time after
carcass addition in days.
7
7
B
6
Protein concentration (mM)
Protein concentration (mM)
A
5
4
3
2
1
0
6
5
4
3
2
1
0
0
2
4
6
8
10
12
14
0
2
4
Time (days)
6
8
10
12
14
Time (days)
Figure S2: Mean protein concentration over time at the high (A) and low (B) dose rates
for each of the treatments: ● Control; ○ 4006; ▼ 7003;  Liquor;  JBL;  4003; Soil
and ◊ Gel 60®. Values represent means (n = 3) ± the standard error of the means.
200
B
A
Amino acid concentration (mM)
Amino acid concentration (mM)
200
150
100
50
0
150
100
50
0
0
2
4
6
8
Time (days)
10
12
14
0
2
4
6
8
10
12
14
Time (days)
Figure S3: The mean concentration of amino acids over time at the high (A) and low (B)
dose rates for each of the treatments: ● Control; ○ 4006; ▼ 7003;  Liquor;  JBL; 
4003; Soil and ◊ Gel 60®. Values represent means (n = 3) ± the standard error of the
means.