MethodsFiber sludgesThe waste fiber

Methods
Fiber sludges
The waste fiber sludges that were used in this study were
kindly provided by two different European mills, one pulp
and paper mill using a sulfate-based process (SAFS) and a
lignocellulosic biorefinery using a sulfite-based process
(SIFS). The characterization and analysis of the feedstocks
and the content of monosaccharides, lignin and ash was
performed by MoRe Research (Örnsköldsvik, Sweden).


Enzymatic hydrolysis
The hydrolysis of the fiber sludges was performed
enzymatically without any prior thermochemical pretreatment.
Initially, 290 g of moist SAFS with a dryweight
content of 51.7% were mixed with 693.8 g of
citrate buffer (0.05 M, pH 5.0), while 343 g of moist
SIFS with a dry-weight content of 43.7% were mixed
with 640.8 g of the citrate buffer. The fiber sludges
were mixed in 2-L shake flasks, the final dry-matter
content was 15% (w/w), and the total content per
shake flask was 1 kg. The enzyme preparation used for
hydrolysis was Cellic CTec2 (Novozymes, Bagsvaerd,
Denmark). The enzyme preparation was added to a
final concentration of 1.6% (w/w) of the reaction mixture,
which corresponded to 10 FPU/g biomass (dry
weight of waste fiber sludge). The flasks were
incubated with orbital shaking (Ecotron, Infors AG,
Bottmingen, Switzerland) at 50°C and 150 revolutions
per minute (rpm) for 48 h. The glucose level during hydrolysis
was monitored using a glucometer (Glucometer
Elite XL, Bayer Healthcare, Leverkusen, Germany).
After hydrolysis, the slurries were centrifuged (Allegra
X-22R, Beckman Coulter, Brea, CA, USA) at 4°C and
4,200 g for 10 min to recover the liquid fractions. The
pH of the liquid fractions was adjusted to 2.0 with sulfuric
acid, and they were then stored in a freezer before
further processing.


Production of bacterial cellulose
Production of BC was performed using Gluconacetobacter
xylinus (Acetobacter xylinus) ATCC 23770 (American Type
Culture Collection, Manassas, VA, USA). A series of
100-mL flasks were filled with 30 mL fiber sludge
hydrolysate and supplemented with 5 g/L yeast extract
and 3 g/L tryptone. Fiber sludge hydrolysates were either
undiluted or diluted two-fold, three-fold and fourfold.
The flasks were autoclaved at 105°C for 30 min in
order to sterilize the growth media. The flasks were
inoculated with 10% (v/v) G. xylinus inoculum, which
was pre-grown for 24 h in a synthetic medium (25 g/L
D-glucose, 5 g/L yeast extract and 3 g/L tryptone,
pH 5.0). The flasks were incubated statically at 30°C for
7 days, after which the yield of BC and the pH value
were measured. Samples of the culture fluid were taken
for analysis of the monosaccharide content.
A second series of experiments consisted of four-fold
diluted fiber sludge hydrolysates and a glucose reference
with similar monosaccharide content. The experiments
were performed as described above but with 100 mL of
medium in 250-mL flasks. The time of incubation was
increased from 7 days in the first experiment to 14 days.
After 14 days of static incubation, the BC membranes
were collected by filtration and were then dried to constant
weight at 105°C. After that, the BC was weighed
for calculation of the yield.
The yield of BC on initial reducing sugar (g/g) was
calculated by dividing the volumetric yield of BC with
the initial concentration of reducing sugar. The yield of
BC on consumed sugar (g/g) was calculated by using the
following equation:
BC yield on consumed sugar ðg=gÞ
¼ BC g ð Þ
Initial reducing sugar  residual sugar ðgÞ
For characterization of BC membranes, the cellulose
pellicle was soaked in a 0.1 M solution of sodium hydroxide
(60 min, 80°C) to remove impurities, such as
culture medium and trapped bacterial cells. A second
wash was performed with deionized water at the same
temperature and for the same period of time. The BC
pellicle was then washed with deionized water until the
pH of washing water was neutral.
Production of cellulase
Enzyme production with Trichoderma reesei Rut C-30 was
performed by using spent hydrolysates obtained after BC
production, and with the addition of 2% (w/v) of the
corresponding dried waste fiber sludge. Reference medium,
which was used as a control, was based on glucose (10 g/L)
with 2% (w/v) additional waste fiber sludge. Separate
cultures with reference medium were used for SAFS and
SIFS. Each of a series of 500-mL flasks contained 100 mL
spent hydrolysate supplemented with 0.1% (w/v) tryptone,
0.05% citric acid, 2% Vogel’s media [23], and 0.015% Tween
80. The flasks containing the media were autoclaved at
110°C for 30 min. The flasks were then inoculated with
10% (v/v) of a suspension of T. reesei pellets from a culture
with glucose-based reference medium that was pre-grown
at 30°C for 36 h. The cultivations were carried out at 28°C
and 160 rpm for the following 8 days.
Enzyme activity assay
The cellulase activity was measured using a reducing
sugar