See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/305222531

Using Paecilomyces variotii in a biorefinery concept to produce single-cell

protein and bioethanol from hardwood spent sulphite liquor

Conference Paper · September 2014

CITATIONS READS

0 308

4 authors:

Tiago Henriques Susana Raquel Pereira

University of Coimbra RAIZ - Instituto de Investigação da Floresta e do Papel
11 PUBLICATIONS 18 CITATIONS 16 PUBLICATIONS 313 CITATIONS

SEE PROFILE SEE PROFILE

Luísa S. Serafim Ana M R B Xavier

University of Aveiro University of Aveiro
88 PUBLICATIONS 4,039 CITATIONS 70 PUBLICATIONS 1,974 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

PhD project View project

i-multiSMART - Innovative multifunctional nanofibrous membranes for removal of mycotoxins in liquid foods View project

All content following this page was uploaded by Tiago Henriques on 21 February 2021.

The user has requested enhancement of the downloaded file.

 Using Paecilomyces variotii in a biorefinery concept to produce single- P-BE47
cell protein and bioethanol from hardwood spent sulphite liquor
T.M. Henriques*, S.R. Pereira, L.S. Serafim, A.M.R.B. Xavier. CICECO, University of Aveiro, Chemistry
Department, Aveiro, Portugal; *tmsh@ua.pt.

This work shows that acidic sulphite pulp industries could use
the fungus Paecilomyces variotii in a biorefinery concept to
valorise hardwood spent sulphite liquor (HSSL), in order to
produce bioethanol after the single-cell protein (SCP)
production, and thus improving their sustainability. P. variotii
was able to use inhibitory compounds present in HSSL to grow,
namely acetic acid, leaving the sugars in the media. The SCP
produced by P. variotii in HSSL is a good candidate for animal
feeding and eventually for human nutrition. Finally, the bio-
detoxification of HSSL showed to be a good approach to allow
bioethanol production by Scheffersomyces stipitis.

Introduction
Hardwood Spent Sulphite Liquor (HSSL) is a
by-product from acidic sulphite pulping of wood
that is very rich in sugars, manly xylose. However,
HSSL also contains a high amount of toxic
compounds, which inhibits microbial growth and
fermentation (1). Nevertheless, as will be shown in
this work, there are strategies that allow the use of
HSSL as a substrate for the production of several
valuable products, on a biorefinery concept (2).
Paecilomyces variotii is a filamentous fungus
able to metabolize some of the toxic compounds
found in HSSL as carbon source (3). For this
reason this fungus can be used in the processing of
HSSL to obtain valuable products, like single cell
protein (SCP) (4) and bioethanol (3).
The P. variotii biomass can be used for SCP
production, which is a high value product. This
fungus was already used to industrially produce
SCP from a softwood SSL (Pekilo process) in
Finland implemented in 1975 (5). However, SCP
production using a HSSL was never attempted.
Furthermore, P. variotii ability to consume toxic
compounds in HSSL, such as acetic acid and
phenolics, can also be used as a detoxification
step. Hence, the HSSL can be bio-transformed into
a more suitable substrate for further bioprocessing
like bioethanol production (3). The yeast
Scheffersomyces stipitis, unlike Saccharomyces
cerevisiae, can ferment both hexoses and pentoses
(6), which is very important since the pentoses are
the major sugars of the HSSL (1). However, S.
stipitis is sensitive to inhibitors present in HSSL
(7), reason why the detoxification step is so
important.
Objectives
This work aimed to show the application of P.
variotii in the production of SCP and bioethanol
from HSSL. In order to achieve these objectives,
two bioprocesses were studied. In the first part of
this work, the production of SCP by P. variotii in a
sequential batch reactor (SBR) was studied. The
final biomass obtained was then analysed to access
protein and DNA content and to disclose the
amino acids (AAs) profile of the protein. In the
second part, the ability of S. stipitis to produce
ethanol in the HSSL previously bio-detoxified
with P. variotii was investigated.
Methods
HSSL, from Eucalyptus globulus, was subjected
to chemical pre-treatment according to Pereira et
al. (2012). This HSSL was then used as a substrate
to grow the fungus P. variotii in a sequential
batch reactor (SBR) with three cycles, where each
cycle ended when the acetic acid concentration
was at a non-inhibitory level for S. stipitis (3).
Two fungal inoculum concentration were tested
(70 and 110 mg of biomass.L-1) (8).
To assess the SCP composition, the DNA was
determined by spectrophotometry (260 and 280
nm) and the protein content by the Biuret method.
The AAs profile was obtained through GC-FID
(9).
The bioethanol production by S. stipitis was
studied in four assays where HSSL was used in 60
% (V/V). The four different media studied were:
chemically-defined media (CDM) (10); HSSL
without detoxification (HSSL); bio-detoxified
HSSL with sugar supplementation
(detox(+)HSSL); and bio-detoxified HSSL without

ChemPort2014 Poster session - Biological Engineering 10-126

sugar supplementation (detox(-)HSSL).
Results
P. variotii proved to be able to consume most of
the acetic acid present in the HSSL (9.06 g.L-1),
leaving in the media most of the xylose (Figure 1).
Although the inoculum increases from 70 mg.L-1
to 110 mg.L-1, the acetic acid uptake rate was very
similar in both assays (0.277g.L-1.day-1 to 0.242
g.L-1.day-1, respectively).
1st cycle 2nd cycle 3th cycle
decrease in ethanol yield and in xylose uptake rate
(0.16 g.g-1 and 0.13 g.L-1.h-1, respectively) in
HSSL without bio-detoxification fermentation,
when compared with CDM test, clearly shows that
the HSSL contains inhibitory compounds for the
yeast. In both assays with the bio-detoxified
HSSL, ethanol yields obtained were similar (0.30
g.g-1 for detox(+)HSSL and 0.31 g.g-1 for detox(-
)HSSL) (Table 2). These results show that the
initial sugar concentration did not affect the
fermentation yield. The ethanol production success
in the bio-detoxified HSSL indicates that P.
variotii has efficiently consumed the inhibitory
compounds present in HSSL. This is also
demonstrated by the increase of xylose uptake rate
from HSSL to detox HSSL (Table 2).

Table 2. Ethanol/substrate yield (YEthanol) and Xylose

uptake rate (rxylose) of the assays with S. stipitis.
YEthanol a rxyloseb
Assay
1st cycle 2nd cycle 3th cycle (g.g-1) (g.L.-1h-1)

CDM 0.35 0.90

detox(+)HSSL 0.30 0.59

detox(-)HSSL 0.31 0.30

Figure 1. HSSL detoxification by P. variotii in SBR

HSSL 0.16 0.13
with three cycles, (A) assay with inoculum of 70 m.L-1
and (B) assay with inoculum of 110 mg.L-1. a
The yield was calculated until the time where the
maximum ethanol concentration was achieved and taking
The biomass produced at the end of the SBR into account xylose and glucose as substrates.
with 70 mg.L-1 of inoculum presented a high b
The xylose uptake rate was calculated until the time
protein content (82.8 %) with a low amount of where the maximum ethanol concentration was achieved.
DNA (1.1 %). Regarding the AAs composition, the
produced protein presented 6 essential AAs (Table Conclusion
1). Therefore the produced SCP can be considered This work demonstrates the feasibility of using
a good candidate for animal feeding (9). the fungus P. variotii in a biorefinery concept to
produce SCP and bioethanol from HSSL. P.
Table 1. Essential amino acids composition of the SCP variotii was able to grow by metabolising
from P. variotii growth in HSSL. inhibitory compounds from HSSL, namely acetic
Essential AAs % (w/w) acid, leaving the sugars in the media. The SCP
produced by P. variotii in HSSL is a good
SCP from HSSL candidate for animal feeding and eventually for
human nutrition, due to the low content of DNA
Histidine 0.00
(1.1 %) and the high amount of protein (82.8 %)
Isoleucine 0.87 which contains 6 essential AAs. The bio-
Leucine 1.87 detoxification of HSSL was a good approach to
Lysine 1.09 allow bioethanol production by S. stipitis since it
Methionine 0.00 allowed an increase in the ethanol yield from 0.16
Phenylalanine 0.65 g.g-1 to 0.31 g.g-1. This work shows that acidic
sulphite pulp industries could integrate the
Tryptophan 0.00
production of bioethanol after the SCP production
Threonine 0.46
improving their sustainability.
Valine 0.80
Regarding the S. stipitis assays (Table 2), the

ChemPort2014 Poster session - Biological Engineering 10-127

 Acknowledgements
This work was funded by FEDER Funds through the Programa Operacional de Competividade – COMPETE and
national funds through FCT – Fundação para a Ciência e a Tecnologia under the project PEST-C/CTM/LA/0011/2013.
Authors also thank FCT for the PhD grant of S. R. Pereira (SFRH / BD / 64552 / 2009).

References
[1] A. P. Marques, D. V. Evtuguin, S. Magina, F. M. L. Amado, A. Prates. Chemical composition of Spent liquors from
acidic magnesium-based Sulphite pulping of Eucalyptus globulus. Journal of Wood Chemistry and Technology.
2009;29(4):322-36.
[2] S. Pereira, D. Fernandes, C. Silva, L. Serafim, A. Xavier, D. Evtyugin. Valorization of a side product of pulp industry
under the concept of Biorefinery. 11th International Chemical and Biological Engineering Conference, September 5-7,
Lisbon, Portugal, 2011.
[3] S. R. Pereira, T. Ivanuša, D. V. Evtuguin, L. S. Serafim, A. M. R. B. Xavier. Biological treatment of eucalypt spent
sulphite liquors: A way to boost the production of second generation bioethanol. Bioresource Technology.
2012;103(1):131-5.
[4] J. B. Almeida e Silva, U.A. Lima, M. E. S. Taqueda, F. G. Guaragna. Use of response surface methodology for
selection of nutrient levels for culturing Paecilomyces variotii in eucalyptus hemicellulosic hydrolyzate. Bioresource
Technology. 2003;87(1):45-50.
[5] K. G. Forss, K. Jokinen, M. Savolainen. Pekilo fermentation of wood hydrolysates. TAPPI Forest Biology. Wood
Chemistry Conference 1977:101-5.
[6] S. R. Pereira, D. J. Portugal-Nunes, D. V. Evtuguin, L. S. Serafim, A. M. R. B Xavier. Advances in ethanol
production from hardwood spent sulphite liquors. Process Biochemistry. 2013;48(2): 272-282.
[7] A. M. R. B. Xavier, M. F. Correia, S. R. Pereira, D. V. Evtuguin. Second-generation bioethanol from eucalypt
sulphite spent liquor. Bioresource Technology. 2010;101(8):2755-61.
[8] A. P. A Tavares, M. S. M. Agapito, M. A. M. Coelho, J. A. Lopes Da Silva, A. Barros-Timmons, J. A. Coutinho, A.
M. R. B. Xavier. Selection and optimization of culture medium for exopolysaccharide production by Coriolus
(Trametes) versicolor. World Journal of Microbiology and Biotechnology. 2005;21(8-9):1499-507.
[9]. S. R. Pereira SR, L. S. Serafim LS, A. M. R. B. Xavier. Production of single cell Protein from eucalypt sulfite spent
liquor by Paecilomyces variotii. Process Biochemistry. 2014. Submited.
[10] C. Verduyn, E. Postma, W. A. Scheffers, J. P. Van Dijken. Effect of benzoic acid on metabolic fluxes in yeasts: A
continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast. 1992;8(7):501-17.

ChemPort2014 Poster session - Biological Engineering 10-128

View publication stats