Journal Pre-proof

Ethanol production from waste pizza by enzymatic hydrolysis and

fermentation

Yanxu Liu (Writing - original draft) (Methodology) (Investigation),

Wei Han (Conceptualization) (Methodology) (Supervision), Xiaobin
Xu (Writing - original draft), Long Chen (Investigation), Junhong
Tang (Supervision) (Writing - review and editing), Pingzhi Hou
(Writing - review and editing)

PII: S1369-703X(20)30043-7
DOI: https://doi.org/10.1016/j.bej.2020.107528
Reference: BEJ 107528

To appear in: Biochemical Engineering Journal

Received Date: 3 November 2019

Revised Date: 24 January 2020
Accepted Date: 4 February 2020

Please cite this article as: Liu Y, Han W, Xu X, Chen L, Tang J, Hou P, Ethanol production from
waste pizza by enzymatic hydrolysis and fermentation, Biochemical Engineering Journal
(2020), doi: https://doi.org/10.1016/j.bej.2020.107528

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© 2020 Published by Elsevier.

 Ethanol production from waste pizza by enzymatic hydrolysis

and fermentation
Yanxu Liua,c, Wei Hana,b,c, Xiaobin Xud,e, Long Chend, Junhong Tanga, Pingzhi
Houd,e
a
College of Materials and Environmental Engineering, Hangzhou Dianzi University,
Hangzhou 310018, China
b
School of Automation, The Belt and Road Information Research Institute, Hangzhou
Dianzi University, Hangzhou, 310018, China

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c
Zhejiang Jinhuanbao Biotechnology Company Limited, Huzhou, 313300, China
d
The Sci-Tech Academic Institute, Hangzhou Dianzi University, Hangzhou, 310018,

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China
e
Hangzhou YanShi S&T Company Limited, Hangzhou, 210018, China
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
Corresponding authors: Wei Han, Tel.: +86 13738020870. E-mail addresses:
hanwei1982@hdu.edu.cn; Junhong Tang, E-mail addresses: dr_tjh@163.com

1
Highlights

 Feasibility of using waste pizza for ethanol fermentation was examined

 Enzymatic hydrolysis of WP by -amylase could be completed within 50 min

 The total power consumed for the whole bio-process was 1.1 kWh

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 The highest ethanol yield of 0.292 g/g WP was obtained

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 This is the first study to report bio-ethanol production from WP

Abstract
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In this work, the feasibility of ethanol production from waste pizza (WP) by
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enzymatic hydrolysis and fermentation was investigated. The effects of enzyme
volumes (0.02-0.08 mL/L) and WP mass ratios (50-160 g/L) on the performance of
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enzymatic hydrolysis and ethanol fermentation were also examined. It was observed
that the lowest enzyme volume (0.02 mL/L) could completely hydrolyze WP with
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mass ratio of 50 g/L. The reducing sugar (RS) increased obviously with increasing of
WP mass ratio from 50 g/L to 160 g/L when the enzyme volume of 0.02 mL/L was
applied. The WP hydrolysate was utilized as substrate for bio-ethanol production with
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the highest cumulative ethanol production of 27.58 g/L and ethanol yield of 0.292 g/g
WP, respectively. To our knowledge, this is the first study to report ethanol production
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from WP by enzymatic hydrolysis and fermentation.

Keywords: Enzymatic hydrolysis; Ethanol fermentation; Mass balance; Waste pizza;

Power consumed

2
1. Introduction
A worldwide interest in alternative sources of energy has been increasing with the
depletion of fossil fuels [1]. Ethanol is nowadays one of the most served alternative
biofuels or gasoline enhancer [2]. Its market demand increased from 2  109 L to 64 
109 L in the last few decades [3]. Bio-ethanol production has been realized from
various raw materials, such as sugar cane, wheat and corn [4,5]. However, sugar cane
is not appropriate for cultivation in the American and European countries because of
the climatic conditions [6]. While utilization of starchy materials (wheat and corn) for
bio-ethanol production has caused considerable debate about its sustainability since

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1/7 people in the world is suffering from lack of food [7]. Instead, the organic solid
waste is considered to be a good option due to its low cost, great availability and no

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relation with food generation [8]. For instance, it was reported that food waste and
waste sludge were produced at approximately 761 million tons per year [9]. Especially,
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food waste is rich in carbon source with carbohydrate as high as 68% of total solid
which is a promising feedstock for bio-ethanol production [10].
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Pizza is one of the most popular fast-food in the world [11]. More than 100 million
tons of pizza was made and 10% of pizza was wasted per year [12,13]. Waste pizza
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(WP) is a complex solid waste containing various components such as starch, protein
and fat [14,15]. Hydrolysis is considered to be the limited-step for bioconversion of
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organic solid waste (including WP) for biofuels production [16,17]. A pretreatment is
needed to hydrolyze the WP and generate fermentable sugars [2]. The pretreatment for
organic solid wastes has been performed in various ways such as chemical
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pretreatment [18], heat treatment [19] and enzymatic hydrolysis [20]. Enzymatic
hydrolysis has lower utility cost than heat treatment and could not encounter the
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corrosion problem compared to chemical pretreatment [21]. And, the enzyme

producing cost is reducing with the development of biotechnology [22]. However,
litter information about ethanol production from WP by enzymatic hydrolysis and
fermentation was found.
Therefore, this study investigated the feasibility of bio-ethanol production from WP
via hydrolysis and fermentation. The effects of enzyme volumes and WP mass ratios
3
on the performance of enzymatic hydrolysis and further ethanol fermentation were
also examined. The detailed schematic flow of ethanol production from WP by
enzymatic hydrolysis and fermentation was illustrated in Fig. 1. It was expected that
the results achieved from this work could supply a potential way for practical
application of ethanol production from WP.

2. Materials and methods

2.1 WP and pre-treatment
WP used in this study was provided by Pizza Hut located in Hangzhou, China. The

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undesirable materials (such as plastic and tissue) contained in the WP was removed by
hand. Then, the WP was transferred into a mechanical mixer to cut into smaller size

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and sieved by 1.5 mm sieve. The WP was not washed or autoclaved prior to be
hydrolyzed.
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2.2 Enzymatic hydrolysis of WP
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The WP hydrolysis was performed at 90 °C and 300 rpm for 150 min in 1 L Duran
bottle (effective volume). The effects of enzyme volumes (P1: 0.02 mL/L, P2: 0.04
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mL/L and P3: 0.08 mL/L) and WP mass ratios (P1: 50 g/L, P4: 100 g/L, P5: 160 g/L)
on the hydrolysis efficiency were investigated, respectively. The commercial enzyme
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used for WP hydrolysis was -amylase (specific activity of 7 × 104 U/mL) which was
provided by Jinhuanbao Co., LTD (Huzhou, China). The pH adjustment was not
applied in this study to try to reduce the cost. The solid fraction was removed by
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centrifuge (8000 rpm, 20 min) and filtration (filter) after hydrolysis. Then, the
obtained WP hydrolysate was used for ethanol fermentation.
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2.3 Bio-ethanol production from WP hydrolysate

The yeast Saccharomyces cerevisiae used for bio-ethanol production was supplied by
the Anqi Yeast Ltd. (Hubei, China). Prior to be used for ethanol fermentation, it was
cultivated in a flask with water and glucose (100:1000:1, w/w) at 30 °C for 30 min to
obtain the yeast solution. Ethanol fermentation was performed in the fermentor with
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working volume of 2.5 L. The obtained WP hydrolysate was added to the fermentor
and then inoculated 10 mL yeast solution. The conditions for ethanol fermentation
were 30 °C and 200 rpm. The pH adjustment and nutritional supplementation were
also not applied.

2.4 Analysis
In the enzymatic hydrolysis of WP and ethanol fermentation, the aqueous phase of
WP mixture was collected to analyze the COD and RS concentrations. Prior to
analysis, the aqueous sample was centrifuged at 10000 rpm with 5 min and then

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filtrated by the 0.45 m filter [23]. COD concentration, protein and carbohydrates
were determined by the Standard Methods [24] while RS was measured using the

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dinitrosalicylic acid method [25]. Ethanol production was analyzed using the gas
chromatography with a capillary column. Nitrogen was used as the carrier gas with
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the flow rate of 1.2 mL/min. The temperatures of column, injection and flame
ionization were 60 °C, 220 °C and 230 °C, respectively. The power (electricity)
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consumed in the enzymatic hydrolysis and fermentation was measured by electricity
meter. All the samples were determined in triplicate and the data provided in this
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study were averages.

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3. Results
3.1 Waste pizza hydrolysis
The characteristics of the WP were described in Table 1. It was observed that the WP
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was mainly composed of carbohydrate, fat and protein. Based on the characteristics
analysis, the total solid of WP was 71.6%. Besides, the WP was rich in carbohydrate
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(34%) which made the WP to be a potential feedstock for ethanol fermentation.

Fig. 2 presented the effect of enzyme volumes (P1: 0.02 mL/L, P2: 0.04 mL/L and
P3: 0.08 mL/L) on the performance of WP hydrolysis. The WP was hydrolyzed by the
-amylase and an increasing trend for COD and RS production from the beginning to
50 min was found. There are no big difference for COD and RS production with
further hydrolysis time. This result was similar to Demirci’s study which indicated
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that the RS production was little change when the hydrolysis time was too long and
higher yield was only achieved at the early stage of hydrolysis [14]. So, the desired
time for WP hydrolysis was 50 min in this study.
It was also observed from Fig. 2 that COD and RS productions with P1-P3 were
similar. This means the least enzyme volume (P1) applied in this study was enough
for WP hydrolysis. Increased enzyme volumes (P2-P3) could not release higher
COD/RS from WP. This result was different from Kim’s study that the rate of
hydrolysis was determined by the enzyme added to the substrate [11]. In our opinion,
the hydrolysis rate will increase with the increasing of enzyme volumes only when the

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enzyme volume was lower than the hydrolysis needed. There is a balance between
enzyme volume and substrate concentration for hydrolysis rate and efficiency. In this

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study, the enzyme volume of 0.02 mL/L (P1) was the best choice for hydrolysis when
the WP mass ratio was 50 g/L.
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Due to better hydrolysis efficiency of 0.02 mL/L (P1) compared to other enzyme
volumes (P2-P3), WP mass ratios (P1: 50 g/L, P4: 100 g/L and P5 160 g/L)
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optimization was further investigated. The COD and RS production in the hydrolysis
using different WP ratios were described in Fig 3. It was observed that with an
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increasing of WP mass ratios from 50 g/L to 160 g/L, the COD and RS increased
significantly and got the highest values of 75.5 g/L and 41.6 g/L, respectively, with
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WP mass ratio of 160 g/L (P5). This result was within expectation since higher WP
mass ratio could provide more nutrients for COD and RS production.
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3.2 Ethanol fermentation from WP hydrolysate

The performances of bio-ethanol production from WP hydrolysate were shown in the
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Fig. 4. There are no big differences of COD variation (Fig. 4a) for all the batches (P1,
P4 and P5) in the ethanol fermentation. This is interesting because the RS
consumption and ethanol production create a good balance for COD variation. The
final COD concentrations for P1, P4 and P5 were 24.3 g/L, 49.8 g/L and 77.1 g/L,
respectively.
The consumption of RS by the yeast and cumulative ethanol production (CEP)
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from WP hydrolysate were described in Fig. 4 (b) and (c), respectively. The RS was
consumed rapidly by the yeast for ethanol production and no lag phase was found for
all the batches (P1, P4 and P5). The CEP increased from 14.58 g/L to 27.58 g/L with
increasing of RS concentration because higher carbon source could be available for
ethanol fermentation. The maximum CEP of 27.58 g/L was obtained at the RS
concentration of 27.32 g/L (P5).
The CEP was described using a modified Gompertz equation (Eq. 1)
E(t) = P exp - exp(Rme/P) ( - t) +1 (1)
where E(t) is CEP (g/L), P is the highest potential ethanol production (g/L), Rm is

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the highest ethanol production rate (g/L/h),  is the lag phase time (h), e is 2.71 and t
is the fermentation time (h). It was observed from Table 2 that the ethanol production

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potential and ethanol production rate increased with increasing of RS concentration.
The highest ethanol production potential of 33.7 g/L and ethanol production rate of
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8.3 g/L/h were achieved at the RS concentration of 27.32 g/L.
Table 3 summarized the performance of CEP from WP hydrolysate by various RS
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concentrations (obtained from different WP mass ratios). Although the highest CEP of
27.6 g/L was achieved at the maximum RS concentration of 27.3 g/L (P5), the
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overload RS concentration on the yeast was found when RS concentration increased

from 11.46 g/L (P1) to 27.3 g/L (P5). The highest RS consumption of 62.5% and
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ethanol yield of 0.292 g/g WP were obtained at batch P1. These results were similar to
our previous study that higher carbon source could lead to the negative effect on the
microorganisms for biofuels production [26].
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4. Discussion
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In order to get the efficient bioconversion of organic solid wastes for biofuels
production, pretreatment is regarded as a key step. Huang examined the
biodegradability of food waste at high pressure and temperature using microwave
irradiation and showed that the solubilization of food waste could be increased
effectively [27]. Uppungundla successfully improved the hydrolysis speed and
conversion efficiency from kitchen waste by acid pretreatment [28]. Although
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physical and chemical pretreatments have been widely used and investigated, the
above methods could consume a great amount of energy or generate inhibitors (such
as furfural) for further ethanol fermentation.
In this study, the power (electricity) consumed for enzymatic hydrolysis was
calculated. It was found in Table 4 that the power consumed for all the batches was
around 1.1-1.3 kWh. As the desired hydrolysis time was 50 min (Fig. 2), the actual
power consumed for WP hydrolysis was 0.4 kWh. The power consumed in the
hydrolysis was mainly used for heating (90 °C). The high temperature is good for
enzyme to attack the starch kernels and get the fermentable sugars easily. And, no

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inhibitory by-products could be generated for further ethanol fermentation compared
to the acid/alkaline pretreatment.

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According to the COD and RS produced in the WP hydrolysate, the COD and RS
yields could be calculated (Table 4). The COD and RS yields for P1-P3 (WP: 50 g/L)
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were around 0.454-0.556 g/g WP and 0.256-0.264 g/g WP, respectively. The COD and
RS yields increased to 0.566 g/g WP and 0.285 g/g WP with an increasing of WP to
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100 g/L (P4), while slightly decreased to 0.472 g/g WP and 0.26 g/g WP when the WP
mass ratio further increased to 160 g/L (P5). Higher nutrients could be obtained with
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an increasing of WP mass ratios from 50 g/L to 160 g/L. However, the higher RS
released from the hydrolysis could inhibit the activity of the enzyme and lead to the
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lower hydrolysis efficiency. Han et al. reported the similar results that the RS yield
decreased from 0.534 g/g wheat flour to 0.439 g/g wheat flour when the wheat flour
mass ratio increased from 2% to 8% (w/v) [29].
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In the ethanol fermentation, the ethanol produced with time and no lag phase was
observed. It was indicated that the WP hydrolysate could be used as feedstock for
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bio-ethanol production without any nutritional supplementation. Fig. 5 described the

mass balance of bio-ethanol production from WP. It was observed that the highest
ethanol yield of 0.292 g/g WP was obtained at the condition of WP mass ratio of 50
g/L and enzyme volume of 0.02 mL/L with total power consumed of 1.1 kWh. This
result demonstrated the potential of ethanol production via enzymatic hydrolysis and
ethanol fermentation from WP. To our knowledge, this is the first study to report
8
bio-ethanol production from WP.

5. Conclusions
Waste pizza could be utilized as feedstock for ethanol production by enzymatic
hydrolysis and fermentation without pH adjustment and any nutritional
supplementation. The enzyme volume of 0.02 mL/L was the best choice for
hydrolysis when the WP mass ratio was 50 g/L. The highest ethanol yield of 0.292 g/g
WP was obtained at the condition of WP mass ratio of 50 g/L and enzyme volume of
0.02 mL/L with total power consumed of 1.1 kWh. The results achieved from this

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work could supply a potential way for practical application of ethanol production
from WP.

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Yanxu Liu: Writing- Original draft preparation, Methodology and Investigation.
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Wei Han: Conceptualization, Methodology and Supervision.
Xiaobin Xu: Writing- Original draft preparation.
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Long Chen: Investigation
Junhong Tang: Supervision and Editing
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Pingzhi Hou: Editing

Declaration of interests
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The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Acknowledgement
This work was supported by Zhejiang Province Key R&D projects (No. 2019C03104)
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and (No. 2018C01031) of Hangzhou Yan Shi Technology Co., Ltd., National Science
Foundation of China (No. 61771178 and 41373121) and Science and technology
foundation of Hangzhou Dianzi University(CXJJ2018018).

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Fig. 1 Schematic flow of bio-ethanol production from waste pizza by hydrolysis and
fermentation.

Fig. 2 Effect of enzyme volumes on the performance of waste pizza hydrolysis. (a)
COD production; (b) RS production.
Fig. 3 COD and RS production in the hydrolysis using different waste pizza
concentrations. (a) COD production; (b) RS production.

Fig. 4 The performances of bio-ethanol production from WP hydrolysate. (a) COD

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variation; (b) RS consumption; (c) Cumulative ethanol production.
Fig. 5 Mass balance for bio-ethanol production from waste pizza (50 g) with enzyme

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volume of 0.02 mL/L (Batch number: P1).

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Fig. 1 Schematic flow of bio-ethanol production from waste pizza by hydrolysis and

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fermentation.

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 40
a
35

30

COD (g/L)
25

20

15

10
5 P1 P2 P3
0

20
b 18

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16
14
RS (g/L)

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12
10
8
6
4
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2 P1 P2 P3

0
0 50 100 150 200
lP

Time (min)

Fig. 2 Effect of enzyme volumes on the performance of waste pizza hydrolysis. (a)
COD production; (b) RS production.
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 90
a
80
70
60
COD (g/L) 50
40
30
20
10 P1 P4 P5
0

45
b 40

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35
30

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RS (g/L)

25
20
15
10
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5 P1 P4 P5
0
0 50 100 150 200
Time (min)
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Fig. 3 COD and RS production in the hydrolysis using different waste pizza
concentrations. (a) COD production; (b) RS production.
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 90
a
80
70
60
COD (g/L) 50
40
30
20
10 P1 P4 P5
0

35
b

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30 P1 P4 P5

25

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RS (g/L)

20

15

10
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5
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0

35
lP

c
30

25
Ethanol (g/L)

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20

15
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10

5 P1 P4 P5
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0
0 20 40 60 80
Time (h)

Fig. 4 The performances of bio-ethanol production from WP hydrolysate. (a) COD

variation; (b) RS consumption; (c) Cumulative ethanol production.
 Waste pizza
(50 g)

Commercial Enzymatic
-amylase hydrolysis
(0.02 mL/L)

WP hydrolysate
(13.2 g/L RS)

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Ethanol yield (0.292

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g/g WP) Ethanol
Power consumed: fermentation
1.1 kWh
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Fig. 5 Mass balance for bio-ethanol production from waste pizza (50 g) with enzyme
volume of 0.02 mL/L (Batch number: P1).
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Table 1 Characteristics of the waste pizza used in this study.

Parameters Contents Units (100 g pizza)

Calorie 382 Kj
Moisture 28.37 %
Protein 13 g
Carbohydrate 34 g
Fat 22 g
Saturated fat 5 g
Sodium 2.35 g

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Fiber 2 g
Sugar 1 g

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Table 2 Gompertz equation constants for bio-ethanol production from WP
hydrolysate.

RS P (g/L) Rm (g/L/h)  (h) R2

concentration
(g/L)
11.46 15.8 5.6 0.8 0.95
23.4 19.8 7.2 1.2 0.93
27.32 33.7 8.3 0.9 0.98

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Table 3 Comparisons of bio-ethanol production from WP hydrolysate hydrolyzed by
various enzyme volumes.

Batch Cumulative RS RS RS Ethanol Power

number ethanol concentration consumed consumption yield consumed
production (g/L) (g/L) rate (%) (g/g (kWh)
(g/L) WP)
P1 14.6 11.46 7.17 62.5 0.292 0.9
P4 18.8 23.4 11.1 47.4 0.188 0.7
P5 27.6 27.3 8.16 29.9 0.173 0.7

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Table 4 Comparisons of waste pizza hydrolysis with various enzyme volumes and
waste pizza concentrations. Average values were shown in the Table.

EV WP COD COD RS RS Power

(mL/L) (g/L) concentration yield concentration yield consumed

(g/L) (g/g (g/L) (g/g (kWh)

WP) WP)

P1 0.02 50 22.7 0.454 13.2 0.264 1.3

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P2 0.04 50 27.8 0.556 12.8 0.256 1.2

P3 0.08 50 26.3 0.526 12.9 0.258 1.2

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P4 0.02 100 56.6 0.566 28.5 0.285 1.2

P5 0.02 160 75.5 0.472 -p41.6 0.26

EV: Enzyme volume; WP: Waste pizza; COD: Chemical oxygen demand; RS:
1.1
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Reducing sugar;
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