Detoxification of phenols in sugarcane syrup for improved ...
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Introduction Results & Discussion Conclusions In industrial fermentations, plant biomass, such as sugarcane syrup, is used to provide the carbon source for the yeast to grow and biosynthesize the desired added-value compounds [1]. Sugarcane syrup contains several polyphenols that may influence the fermentation process, exerting antimicrobial activity. Ferulic acid is being mentioned as one of the most toxic phenolic compounds which is present in the syrup [2]. Reducing the polyphenol concentration may help to improve the yeast growth rate, yield or productivity [3]. Detoxification of syrup through using activated charcoals or specific bead resins has the potential to drastically reduce phenols content [4, 5]. This work aims at extracting the phenols present in sugarcane syrup by using solid adsorbents to assess the effect of phenols removal on yeast fermentation. SYRUP CHARACTERIZATION References Charcoal (Carbone Superattivo CP) was more effective than bead resins in the adsorption of polyphenols from sugarcane syrup. Agitation speed and time were tested, and it did not influence phenols detoxification by charcoal. Very high detoxification was reached (up to 96.42 %, measured by LC-MSQTOF) using 100 g of charcoal per L of syrup. Gentisic acid, hydroxybenzoic acid and hydroxybenzoic-4-B-glucoside remained in the syrup after detoxification. Planification Alchemy Project (PT2020/POCI n.º 27578), co-financed by Portugal 2020 under the Operational Programme for Competitiveness and Internationalization through the European Regional Development Fund (FEDER). Table 1 – Characterization of sugarcane syrup used in industrial fermentations. OPTIMIZATION OF SYRUP DETOXIFICATION Figure 2 – Optimization of sugarcane syrup detoxification using charcoal Proenol: pictures of syrup in mixture with charcoal before (A) and after detoxification (B); total phenols, according to Folin Ciocalteu’s method, and phenol detoxification percentage throughout incubation time (C). To improve detoxification, the concentration of charcoal was increased from 50 to 100 g/L of syrup (Fig. 3). With the highest charcoal concentration, 96.42 % of detoxification was reached. The compounds still remaining in syrup were gentisic acid, hydroxybenzoic acid and hydroxybenzoic-4-B-glucoside at 0.18, 0.41 and 1.28 mg/L, respectively, according to LC-MSQTOF. Figure 3 – Quantification of total phenols, according to Folin Ciocalteu’s methodology (A) and sum of individual quantification by LC-MSQTOF (C). Percentage of polyphenols detoxification (%), according to Folin Ciocalteu’s (B) and LC-MSQTOF methodologies (D). Higher number of asterisks represents higher statistically significant difference. SYRUP DETOXIFICATION WITH DIFFERENT ADSORBENTS A large screening was performed to assess the efficacy of different materials in the detoxification of phenolic compounds from sugarcane syrup. A clear difference between the charcoals and the bead resins was observed: the resins were less effective, presenting lower phenols adsorption capacity. The material with the best performance (highest resulting detoxification) was the charcoal “Carbone Superattivo CP” (Proenol, Portugal). This was selected as the best adsorbent for polyphenols and used for the following studies. Detoxification of phenols in sugarcane syrup for improved yeast fermentation 1 Amyris BioProducts Portugal, Unipessoal, Lda. Rua Diogo Botelho 1327, 4169-005 Porto, Portugal 2 Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal E-mail: [email protected]; [email protected] Luís Carlos Carvalho 1,2 , Ana Oliveira 2 , Erdem Çarşanba 1,2 , Manuela Pintado 2 & Carla Oliveira 2 0 10 20 30 40 50 60 70 80 90 Activated Charcoal Charcoal Proenol Dianion HP-20 LXA 817 LXA 858 AD-200 Amberlite XAD Sepabeads SP-207 Detoxification (%) Figure 1 – Percentage (%) of detoxification of total phenols, according to Folin Ciocalteus’s methodology, obtained for each material tested. Two charcoals (blue) and six bead shaped resins (green) were tested. Parameter Concentration Total reducing sugars 714.8 g/L Total phenols (Folin Ciocalteu’s) 2080 ± 119 mg/L Sum of identified phenols (LC- MSQTOF) 52.35 ± 1.05 mg/L Hydroxybenzoic acids 13.70 ± 0.27 mg/L Hydroxycinnamic acids 27.07 ± 0.41 mg/L Flavones 11.59 ± 0.12 mg/L pH 5.58 ± 0.01 Total reducing sugars (TRS) – calorimetric determination Total phenolic content - Folin Ciocalteu’s and LC-MSQTOF Screening for efficacy of different adsorbents Syrup Characterization Syrup Detoxification Optimization of detoxification Erlenmeyer flask fermentation Bioreactor fermentation Fermentation • Baffled Erlenmeyer flasks • 50 g of adsorbent/ L of syrup • 22 h of agitation at 100 rpm at room temperature • Baffled Erlenmeyer flasks • 6 h of agitation at 60 rpm • Saccharomyces cerevisiae strain producing industrially relevant terpene • 30 ºC and 200 rpm • Growth rate and oxidative stress • Simulation of industrial conditions • Assessment of potential scale-up Separation • Centrifugation at 16 345 g • Filtration with 2-layer filter paper type I Bioproduct quantification • Gas chromatography Adsorption was conducted by reducing the agitation time and speed (Fig. 2-C). After 4 hours of agitation at 60 rpm, the polyphenols detoxification reached maximum value of 72.73 %. The detoxification process will be further optimized to obtain complete removal of phenols and minimize charcoal usage. Shake-flask fermentations will be performed to evaluate yeast performance in phenols-free syrup, as compared to its performance in non-purified syrup. Bioreactor fermentations, simulating industrial conditions, will be implemented to validate the results found in flasks, and the potential for scaling-up syrup detoxification will be assessed. Future Work 0 500 1000 1500 2000 2500 0 10 20 30 40 50 60 70 80 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Total phenols (mg/L) Detoxification (%) Time (h) Detoxification Total phenols A B C 0 10 20 30 40 50 60 70 80 90 100 50 100 Detoxification - Folin (%) Charcoal concentration (g/L) 0 500 1000 1500 2000 2500 0 50 100 Total phenols - Folin (mg/L) Charcoal concentration (g/L) 0 10 20 30 40 50 60 0 50 100 Total phenols – LC-MS (mg/L) Charcoal concentration (g/L) 0 10 20 30 40 50 60 70 80 90 100 50 100 Detoxification – LC-MS (%) Charcoal concentration (g/L) A B C D * ** *** * ** ** * *** * ** [1] Cunha, et al. (2019) Applied Microbiology and Biotechnology, 103, 159-175. [2] Adeboye, et al. (2014) AMB Express, 4, 46. [3] Wang, et al. (2016) BMC biotechnology, 16, 31-31. [4] Vallejos, et al. (2016) Industrial Crops and Products, 91, 161-169. [5] Yu and Christopher (2017) Fuel, 203, 187-196. Characterization of sugarcane syrup, used in industrial fermentations, was performed.
Transcript of Detoxification of phenols in sugarcane syrup for improved ...
Intr
od
ucti
on
Resu
lts
& D
iscuss
ion
Co
nclu
sio
ns
In industrial fermentations, plant biomass, such as sugarcane syrup, is used to
provide the carbon source for the yeast to grow and biosynthesize the desired
added-value compounds [1].
Sugarcane syrup contains several polyphenols that may influence the fermentation
process, exerting antimicrobial activity. Ferulic acid is being mentioned as one of
the most toxic phenolic compounds which is present in the syrup [2].
Reducing the polyphenol concentration may help to improve the yeast growth
rate, yield or productivity [3].
Detoxification of syrup through using activated charcoals or specific bead resins
has the potential to drastically reduce phenols content [4, 5].
This work aims at extracting the phenols present in sugarcane syrup by using solid
adsorbents to assess the effect of phenols removal on yeast fermentation.
SYRUP CHARACTERIZATION
Refe
ren
ces
Charcoal (Carbone Superattivo CP) was more effective than bead resins in the
adsorption of polyphenols from sugarcane syrup.
Agitation speed and time were tested, and it did not influence phenols
detoxification by charcoal.
Very high detoxification was reached (up to 96.42 %, measured by LC-MSQTOF)
using 100 g of charcoal per L of syrup.
Gentisic acid, hydroxybenzoic acid and hydroxybenzoic-4-B-glucoside
remained in the syrup after detoxification.
Pla
nif
ica
tio
n
Alchemy Project (PT2020/POCI n.º 27578), co-financed by Portugal 2020 under the Operational Programme for Competitiveness and Internationalization through the European Regional Development Fund (FEDER).
Table 1 – Characterization of sugarcane syrup used in industrial fermentations.
OPTIMIZATION OF SYRUP DETOXIFICATION
Figure 2 – Optimization of sugarcane syrup detoxification using charcoal Proenol: pictures of syrup in
mixture with charcoal before (A) and after detoxification (B); total phenols, according to Folin Ciocalteu’s
method, and phenol detoxification percentage throughout incubation time (C).
To improve detoxification, the concentration of charcoal was increased from 50 to 100
g/L of syrup (Fig. 3). With the highest charcoal concentration, 96.42 % of detoxification
was reached. The compounds still remaining in syrup were gentisic acid,
hydroxybenzoic acid and hydroxybenzoic-4-B-glucoside at 0.18, 0.41 and 1.28 mg/L,
respectively, according to LC-MSQTOF.
Figure 3 – Quantification of total phenols, according to Folin Ciocalteu’s methodology (A) and sum of
individual quantification by LC-MSQTOF (C). Percentage of polyphenols detoxification (%), according to
Folin Ciocalteu’s (B) and LC-MSQTOF methodologies (D). Higher number of asterisks represents higher
statistically significant difference.
SYRUP DETOXIFICATION WITH DIFFERENT ADSORBENTS
A large screening was performed to assess the efficacy of different materials in the
detoxification of phenolic compounds from sugarcane syrup.
A clear difference between the charcoals and the bead resins was observed: the resins
were less effective, presenting lower phenols adsorption capacity.
The material with the best performance (highest resulting detoxification) was the
charcoal “Carbone Superattivo CP” (Proenol, Portugal). This was selected as the best
adsorbent for polyphenols and used for the following studies.
Detoxification of phenols in sugarcane syrup for improved yeast fermentation
1 Amyris BioProducts Portugal, Unipessoal, Lda. Rua Diogo Botelho 1327, 4169-005 Porto, Portugal
2 Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de
Biotecnologia, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal
E-mail: [email protected]; [email protected]
Luís Carlos Carvalho1,2, Ana Oliveira2, Erdem Çarşanba1,2, Manuela Pintado2 &
Carla Oliveira2
0
10
20
30
40
50
60
70
80
90
ActivatedCharcoal
CharcoalProenol
DianionHP-20
LXA 817 LXA 858 AD-200 AmberliteXAD
SepabeadsSP-207
Deto
xific
ation (
%)
Figure 1 – Percentage (%) of detoxification of total phenols, according to Folin Ciocalteus’s methodology,
obtained for each material tested. Two charcoals (blue) and six bead shaped resins (green) were tested.
Parameter Concentration
Total reducing sugars 714.8 g/L
Total phenols (Folin Ciocalteu’s) 2080 ± 119 mg/L
Sum of identified phenols (LC-
MSQTOF)
52.35 ± 1.05
mg/L
Hydroxybenzoic acids 13.70 ± 0.27 mg/L
Hydroxycinnamic acids 27.07 ± 0.41 mg/L
Flavones 11.59 ± 0.12 mg/L
pH 5.58 ± 0.01
Total reducing sugars (TRS) – calorimetric determinationTotal phenolic content - Folin Ciocalteu’s and LC-MSQTOF
Screening for efficacy of different adsorbents
Syrup Characterization
Syrup Detoxification
Optimization of detoxification
Erlenmeyer flask fermentation
Bioreactor fermentation
Fermentation
• Baffled Erlenmeyer flasks
• 50 g of adsorbent/ L of syrup
• 22 h of agitation at 100 rpm at
room temperature
• Baffled Erlenmeyer flasks
• 6 h of agitation at 60 rpm
• Saccharomyces cerevisiae strain
producing industrially relevant
terpene
• 30 ºC and 200 rpm
• Growth rate and oxidative stress
• Simulation of industrial
conditions
• Assessment of potential
scale-up
Separation
• Centrifugation
at 16 345 g
• Filtration with
2-layer filter
paper type I
Bioproduct quantification
• Gas chromatography
Adsorption was conducted by reducing the agitation time and speed (Fig. 2-C). After 4
hours of agitation at 60 rpm, the polyphenols detoxification reached maximum value of
72.73 %.
The detoxification process will be further optimized to obtain complete
removal of phenols and minimize charcoal usage.
Shake-flask fermentations will be performed to evaluate yeast performance
in phenols-free syrup, as compared to its performance in non-purified syrup.
Bioreactor fermentations, simulating industrial conditions, will be
implemented to validate the results found in flasks, and the potential for
scaling-up syrup detoxification will be assessed.Fu
ture
Wo
rk
0
500
1000
1500
2000
2500
0
10
20
30
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0.0 1.0 2.0 3.0 4.0 5.0 6.0
Tota
l ph
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ols
(m
g/L
)
Det
oxi
fica
tio
n (
%)
Time (h)Detoxification Total phenols
A BC
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Charcoal concentration (g/L)
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Charcoal concentration (g/L)
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Tota
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S (m
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Charcoal concentration (g/L)
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50 100
Det
oxi
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S (%
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Charcoal concentration (g/L)
A B C D*
**
***
*
**
**
*
***
*
**
[1] Cunha, et al. (2019) Applied Microbiology and Biotechnology, 103, 159-175. [2] Adeboye, et al. (2014) AMB Express, 4, 46. [3] Wang, et al. (2016) BMC biotechnology, 16, 31-31.
[4] Vallejos, et al. (2016) Industrial Crops and Products, 91, 161-169. [5] Yu and Christopher (2017) Fuel, 203, 187-196.
Characterization of sugarcane syrup, used in industrial fermentations, was performed.
