# Innovative microscale workflow from fungi cultures to Cell Wall-Degrading Enzyme screening

Roxane Raulo, Egon Heuson, Ali Siah, Vincent Phalip, Rénato Froidevaux

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Roxane Raulo, Egon Heuson, Ali Siah, Vincent Phalip, Rénato Froidevaux. Innovative microscale workflow from fungi cultures to Cell Wall-Degrading Enzyme screening. Microbial Biotechnology, 2019, 12 (6), pp.1286 - 1292. 10.1111/1751-7915.13405. hal-02624700

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Brief report

Innovative microscale workflow from fungi cultures to
Cell Wall-Degrading Enzyme screening

Roxane Raulo, ID Egon Heuson, Ali Siah, Vincent Phalip and Renato Froidevaux* ID

EA 7394 - ICV - Institut Charles Viollette, Univ. Lille 1, ISA, INRA, Univ. Artois, Univ. Littoral Côte d'Opale, F-59000 Lille, France.

suitable for HT CWDE production thanks to its pipet-
ting platform compatibility. Also, the results showed
that the enzymatic activities measured were the
same when doing the assay manually or using the
automated method.

Summary

This study aimed at developing a complete miniatur-
ized high-throughput screening workflow for the
evaluation of the Cell Wall-Degrading Enzyme
(CWDE) activities produced by any fungal strain
directly cultivated on raw feedstock in a submerged
manner. In this study, wheat straw was selected as
model substrate as it represents an important car-
bon source but yet poorly valorised to yield high
added value products. Fungi were grown in a micro-
bioreactor in a high-throughput (HT) way to replace
the fastidious shaking flask cultivations. Both
approaches were compared in order to validate our
new methodology. The range of CWDE activities pro-
duced from the cultures was assayed using AZO-
died and pNP-linked substrates in an SBS plate for-
mat using a Biomek FXp pipetting platform. As high-
lighted in this study, it was shown that the CWDE
activities gathered from the microbioreactor cultiva-
tions were similar or higher to those obtained from
shake flasks cultures, with a lower standard devia-
tion on the measured values, making this new
method much faster than the traditional one and

Introduction

Enzymes secreted by filamentous fungi have a key role
in the degradation of the most abundant biopolymers
found in nature, that is cellulose and hemicelluloses.
These enzymes (Cell Wall-Degrading Enzymes, CWDE)
are of great interest in the industrial conversion of ligno-
cellulosic substrates into component sugars, which then
serve as substrates for the synthesis of biofuels and
other highly valuable platform molecules. However, the
industrial conversion of lignocellulosic biomasses to such
monomers faces two major bottlenecks: (i) the plant cell
wall is highly recalcitrant to enzymatic degradation due
to the presence of lignin which reduces enzyme accessi-
bility to cellulose and hemicelluloses and (ii) a lack of
high-throughput methods to rapidly screen for the enzy-
matic activities associated to these degradation capabili-
ties in plant-degrading strains.

Fungi are the predominant source of enzymes cur-
rently being used on an industrial scale for the
conversion of lignocellulosic biomass to platform mole-
cules (Archer, 2000; Sims et al., 2010; Gusakov, 2011).
Improving the efficiency of enzymatic saccharification
has been an active area of research during the last dec-
ade, with efforts dedicated towards the discovery and
characterization of novel saccharolytic enzymes (Phalip
et al., 2005; Carapito et al., 2009; Banerjee et al., 2010),
to be used in biorefineries. A biorefinery is a facility that
integrates biomass conversion processes and equipment
to produce fuels, power and value-added chemicals from
biomass. As refineries, biorefineries can provide multi-
ples chemicals by fractioning the lignocellulosic biomass
into multiple intermediates that can be further converted
into value-added products (Menon and Rao, 2012).The
past few years have seen a strong increase in research
topics aiming at the isolation and development of novel
fungal enzyme mixtures, which can be tailored to

Received 18 January, 2019; accepted 17 March, 2019.

*For correspondence. E-mail renato.froidevaux@univ-lille.fr; Tel. +33 (0)3 20 41 75 66; Fax +33 (0)3 28 76 73 56.

Microb Biotechnol (2019) 12(6), 1286–1292

doi:10.1111/1751-7915.13405

Funding Information

This work was supported by the Alibiotech project that is financed
by the European Union, the French State and the French Region of
Hauts-de-France. The REALCAT platform is benefiting from a
Governmental subvention administrated by the French National
Research Agency (ANR) within the frame of the 'Future
Investments' program (PIA), with the contractual reference 'ANR-11-
EQPX-0037'. The Hauts-de-France Region and the FEDER as well
as the Centrale Innovation Fondation are thanked for their financial
contribution to the acquisition of the equipment of the platform.
efficiently degrade recalcitrant lignocellulose into mono-
meric sugars with the help of mild or no thermochemical
pretreatment (Berrin et al., 2012; Couturier et al., 2012).
Currently, functional screening of fungi is associated with
low throughput and prohibitive costs, which severely lim-
its the discovery of novel enzymatic activities and better
CWDE producing strains. Fermentation experiments in
shake flasks, bench-scale bioreactors, or a combination
of both, are the conventional methods for both fungi
screening and fermentation optimization. However, the
screening use of these two methods is limited to a small
number of strains that can be tested in parallel, as well
as the large amount of substrate (feedstock), cultivation
medium, sterilization steps, etc. that require such manip-
ulations. Of course, several HT small scale cultivation
approaches have already been designed and have pro-
ven their efficiency to seek enzyme activities produced
by microorganisms. Among them, the recently released
microbioreactor device from M2PLabs was extensively
used for such application, helping to greatly speed up
the cultivation phase. However, when it comes to raw
feedstocks as substrates, with filamentous fungi in sub-
merged fermentation, no use of such device has been
reported. Developing a screening methodology that
would allow for a larger number of cultivations to be per-
formed in parallel directly on raw feedstock, while retain-
ing conditions that can be easily controlled upon scale-
up, would consequently greatly reduce the development
time and costs of CWDE cocktails from filamentous
fungi, that could later be used to improve the biorefiner-
ies' saccharification processes.

In this study, we present a novel and original
approach to screen and select CWDE cocktails from two
different fungal strains in a semi-automated workflow

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Enzymatic activities measured from the supernatants of *Penicillium chrysogenum* cultures grown on wheat straw for 7 days using the pNP-glucopyranoside assay. The assays were done manually and using the Biomek FXP pipetting platform. Cultivation supernatants obtained from the microbioreactor were assayed using 4-Nitrophenyl-β-D-glucopyranoside (pNP-glucopyranoside, Megazyme) substrate. In a 96 U-well plate, 30 µl of the culture supernatants were incubated with 150 µl of 0.5% (w/v) pNP substrate solution, 75 µl of 100 mM sodium acetate buffer pH 5.0, completed with water up to a total volume of 300 µl. The reaction mixtures were incubated at room temperature for 2 h with 5 min of initial shaking. The reactions were then stopped by adding 50 µl of the reaction mixture to 200 µl of sodium phosphate solution pH 12 (2% w/v). A total of 100 µl of the supernatants were transferred to a flat-bottom 96-well reading plate and the optical densities were measured at 405 nm with a plate reader (SpectraMax i3, Molecular Devices, USA). All experiments were conducted in biological triplicates. Statistical analysis performed did not show any differences between the two conditions.</figcaption></figure>

(Fig. 1). The selected strains are *Penicillium chrysogenum*, a saprophytic species that has not been extensively studied for feedstock degradation, and

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Flow chart of the integrated approach developed for this work with the implementation of high-throughput (HT) filamentous fungi cultures on wheat straw using the microfermentor microbioreactor followed by HT screening of Cell Wall-Degrading Enzymes (CWDE) activities using the Biomek FXP (Beckman Coulter, USA) pipetting station. The programming parameters for the BioLector (M2P-Labs, Germany) and the detailed steps of the Biomek FXP program are described in Appendix S1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Plates layouts for the microbioreactor cultures as well as the enzymatic assays. A. Cultures in the microbioreactor were done in triplicates (column) for both fungi *Penicillium chrysogenum* and *Zymoseptoria tritici*. Cultures were carried out for 4 (D4) and 7 days (D7) in minimal medium M3 supplemented with 1% wheat straw. For cultures of *P. chrysogenum* and *Z. tritici* in shake flasks, 5.10⁵ spores per ml in suspension were inoculated into 250-ml sterile Erlenmeyer flasks containing 100 ml M3 medium (Mitchell *et al.*, 1997), supplemented with 1% milled wheat straw (w/v), and incubated for 7 days at 25°C on a shaker (Infors HT Multitron standard, Switzerland) set to 150 r.p.m. Supernatants were manually collected at day 0, day 4 and day 7 after inoculation for enzymatic analysis. For cultures carried out in the BioLector® microbioreactor (M2PLabs, Germany), a FlowerPlate MTP-48-B (without optodes) was used as reactor. To lower evaporation and maintain the plate sterility, a low evaporation breathing foil F-GPR48-10 was set on top of the plate after filling. Each of the 48 wells was filled with 1.4 ml M3 medium (Mitchell *et al.*, 1997) containing 1% milled wheat straw (w/v). The wells were inoculated at 5.10⁵ spores per ml and incubated for 7 days at 25°C at 800 r.p.m. Supernatants were manually collected at day 0, day 4 and day 7 after inoculation for enzymatic analysis by collecting the full content of the well. Feedstock remaining debris and fungi cells were removed from the supernatant through a filtration step using a 0.22 µm syringe filter. The enzymes activities were measured according to the protocol described previously. All experiments were conducted in biological triplicates. Negative control for both substrates was included. B. Enzymatic assays plate layout and results.</figcaption></figure>

*Zymoseptoria tritici*, the causal agent of wheat blotch. The approach is based on the use of two HT robotic equipment, a Biomek FXP pipetting platform from Beckman Coulter, and the microbioreactor from M2PLabs. This study is the first report of the use of the microbioreactor for the implementation of high-throughput filamentous fungi cultures on complex biomasses followed by the high-throughput screening of enzyme activities. This approach is greatly speeding up the screening of fungal CWDE activities by scaling down the whole process to less than 2 ml fermentations. Therefore, with the workflow, we expect to increase the likelihood of discovering interesting strains and enzymes for industrial

applications that can directly be used on raw feedstocks
in biorefineries.

**Results and discussion**

*Automated enzymatic assaying validation*

The automated enzymatic assay results were validated
by comparing the relative activity measured when plates
were prepared and read manually with the values
obtained after the semi-automated method. It is noteworthy
to precise that, for the semi-automated method, all
the pipetting action were performed by the Biomek plat-
form. The only manual step was the centrifugation step.
<table><caption>**Table 1.** List of the enzymatic assays used in this study</caption><thead><tr><th>Test name</th><th>Activity tested</th><th>Reference manufacturer</th></tr></thead><tbody><tr><td>AZO-xylan (Birchwood)</td><td>endo-1,4-β-D-xylanase</td><td>Megazyme S-AXBP</td></tr><tr><td>AZO-cellulase</td><td>endo-1,4-β-D-glucanase</td><td>Megazyme S-ACMC</td></tr><tr><td>AZO-rhamnogalacturonan</td><td>Rhamnogalacturonan lyase</td><td>Megazyme S-AZRH</td></tr><tr><td>AZO-barley glucan</td><td>β-glucanase</td><td>Megazyme S-ABG100</td></tr><tr><td>pNP-glucopyranoside</td><td>exo-1,4-β-D-glucanase</td><td>Megazyme O-PNPBG</td></tr><tr><td>pNP-xylopyranoside</td><td>exo-1,4-β-D-xylanase</td><td>Megazyme O-PNPX</td></tr></tbody></table>

Both methods showed similar results with 13.8 ± 0.48 10⁻³ mUI ml⁻¹ and 13.1 ± 0.42 10⁻³ mUI ml⁻¹, respectively, measured with the pNP-glucopyranoside assay

when performed manually and with the pipetting station, using wheat straw as substrate and *P. chrysogenum* as fungal strain after 7 days of incubation (Fig. 2).

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Enzyme activities measured using AZO-substrates and pNP-linked substrates after 4 (D4) and 7 days (D7) of cultures in the microbioreactor for *Penicillium chrysogenum* and *Zymoseptoria tritici*. Enzyme activity (A) on AZO-xylan, (B) on AZO-cellulose, (C) on AZO-rhamnogalacturonan, (D) on AZO-barley glucan, (E) on pNP-glucopyranoside and (F) on pNP-xylopyranoside. In a 96 U-well plate, 30 µl of the culture supernatants were incubated with 150 µl of 0.5% (w/v) of the substrate solution, 75 µl of 100 mM sodium acetate buffer pH 5.0, completed with water up to a total volume of 300 µl. The reaction mixtures were incubated at room temperature for 2 h with 5 min of initial shaking. The reactions were then stopped by adding 50 µl of the reaction mixture to 200 µl of ethanol (95% v/v) (AZO-xylan, AZO-cellulose, AZO-rhamnogalacturonan and AZO-barley glucan) or sodium phosphate solution pH 12 (2% w/v) (pNP-glucopyranoside and pNP-xylopyranoside). The precipitated remaining AZO-substrates were removed by centrifugation at 2120 g for 10 min. A total of 100 µl of the supernatants were transferred to a flat-bottom 96-well reading plate and the optical densities were measured at 590 nm and 405 nm, AZO-substrates and pNP-substrates, respectively, with a plate reader (SpectraMax i3, Molecular Devices, USA). All experiments were conducted in biological triplicates. A two-way ANOVA test was performed. * indicates a significant difference within one condition (e.g. D4) when the *P. chrysogenum* and the *Z. tritici* strains were compared (*P* < 0.05).</figcaption></figure>
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**Fig. 5.** Enzyme activities measured using AZO-substrates and pNP-linked substrates after 7 days of cultures in shake flask cultures and in the microbioreactor for *Zymoseptoria tritici*. Enzyme activity (A) on AZO-xylan, (B) on AZO-cellulose, (C) on AZO-rhamnogalacturonan, (D) on AZO-barley glucan, (E) on pNP-glucopyranoside and (F) on pNP-xylopyranoside. For cultures of *P. chrysogenum* and *Z. tritici* in shake flasks, spores were washed with 0.01% (v/v) Tween-80 solution and 5.10⁵ spores per ml in suspension were inoculated into 250 ml sterile Erlenmeyer flasks containing 100 ml M3 medium (Mitchell *et al.*, 1997), supplemented with 1% milled wheat straw (w/v), and incubated for 7 days at 25°C on a shaker (Infors HT Multitron standard, Switzerland) set to 150 r.p.m. Media were autoclaved at 110°C for 30 min instead of 121°C to limit the Maillard reaction. Supernatants were manually collected at day 0, day 4 and day 7 after inoculation for enzymatic analysis. Feedstock remaining debris and fungi cells were removed from the supernatant through a filtration step using a 0.22 µm syringe filter. All experiments were conducted in biological triplicates. The enzymes activities were measured according to the protocol described previously. A t-test was performed for statistical analysis. * indicates a significant difference between the activity measured in the shake flask and in the microbioreactor (*P* < 0.05).

*Cell Wall-Degrading Enzyme activities gathered from fungi*

The capability of the strains used in this study to degrade wheat straw was assayed using a microbioreactor for the implementation of high-throughput filamentous fungi cultures on complex biomasses followed by the high-throughput screening of enzyme activities (Fig. 3).

Enzymatic activities were assayed after both 4 and 7 days of cultures on wheat straw of *P. chrysogenum* and *Z. tritici*. For all the enzymatic assays tested, an increase in enzymatic activities was observed between the D4 and D7 time points (Fig. 4). Using this semi-automated method, the tests assayed (Table 1) were able to discriminate for specific activities for each fungus. The AZO-cellulose test showed higher activities
measurements for *Z. tritici* while the other tests showed that *P. chrysogenum* was more efficient at degrading these substrates. In addition, the method allowed the measurement of low enzyme activities, the lowest being 0.18 10⁻³ mUI ml⁻¹ for *P. chrysogenum* on the pNP-xylopyranoside substrate. These results, and the sensitivity of the assays, demonstrated the potential for the developed method to be used for HT screening of CWDEs on complex substrates.

*Performance comparison between the microbioreactor and the flask cultures*

The comparison in enzymatic activities measured in shake flask cultures compared to the one obtained using the microbioreactor was performed with the *Z. tritici* strain over a time course of 7 days. When comparing the data obtained from both the cultures in shake flasks and in the microbioreactor, the trend in CWDE activities measured was the same with the 5 AZO-substrates, all along the culture. For instance, at day 7, when considering the AZO-xylan substrate, the same levels of enzyme activities were measured for two types of culture, with 1.06 10⁻³ mUI ml⁻¹ and 1.37 10⁻³ mUI ml⁻¹ measured in the shake flask cultures and in the microbioreactor respectively (Fig. 5). The most significant results were obtained for the pNP-linked substrates, with an increase in the enzyme activities measured for the two types of pNP-substrates in the microbioreactor which may be due to a better oxygen transfer rate in the microbioreactor (Fig. 5). These results highlight the potential of the described method for HT screening as the cultures volume was reduced by a factor 70 while allowing for differential enzyme activities measurement. In addition, HT screening of the enzyme activities was also achieved with the use of the Beckman Coulter robot.

Conclusion

When designing an industrial fermentation, a large num-
ber of experiments are required to respectively select a
strain, improve the cultivation medium and optimize the
fermentation procedure, measure the enzymes activities
and finally select the most efficient strain for the degra-
dation of a specific biomass. The limitations of shake
flasks cultures like the inability to control parameters,
other than agitation rate and temperature, usually means
that the scale-up to the bioreactor scale is rather compli-
cated as it is neglecting, for instance, the effect of oxy-
gen supply on growth and the metabolite production
during fermentation. For the first time, these results high-
lighted the potential of the microbioreactor to screen fila-
mentous fungi species on raw feedstocks since similar

and higher CWDE activities were observed from the
microbioreactor cultures compared to the shake flask
ones. These cultivations were done directly using shred-
ded wheat straw as raw substrate in a submerged man-
ner, proving the ability of the equipment to run accurate
cultivations on such complex material with some advan-
tages compared to the current flask cultures method (i.e.
more conditions screened, less amount of substrate
needed, better activities as shown in Fig. 4). In addition,
the scale-up from small scale to large scale bioreactor is
often more predictable than from shake flasks to biore-
actor (Rohe et al., 2012). Overall, the differences in
activities measured in this work were strong enough to
discriminate between the screened strains with different
responses obtained depending on the substrate
assayed. Using this tool, an increased number of strains
and parameters will be tested in a close future, setting
this workflow as an efficient starting point for the discov-
ery of fungal CWDE cocktails that could later be scaled
up to an industrial production level for biorefineries pur-
pose. This method is currently being upgraded as a fully
automated process with the sampling of the super-
natants done directly in the wells using a Beckman Coul-
ter robot (Biomek NX® with a BioLector Pro® integrated
to the deck).

Acknowledgements

This work was supported by the Alibiotech project that is
financed by the European Union, the French State and
the French Region of Hauts-de-France. The REALCAT
platform is benefiting from a Governmental subvention
administrated by the French National Research Agency
(ANR) within the frame of the ‘Future Investments’ pro-
gram (PIA), with the contractual reference ‘ANR-11-
EQPX-0037’. The Hauts-de-France Region and the
FEDER as well as the Centrale Innovation Fondation are
thanked for their financial contribution to the acquisition
of the equipment of the platform.

Conflict of interest

None declared.

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**Supporting information**

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Appendix S1.** Fungal strains cultivation in the BioLector.