Available online at www.sciencedirect.

com

ScienceDirect
Availableonline
Available onlineatatwww.sciencedirect.com
www.sciencedirect.com
Energy Procedia 00 (2017) 000–000

ScienceDirect
ScienceDirect
www.elsevier.com/locate/procedia

Energy
EnergyProcedia
Procedia136 (2017) 000–000
00 (2017) 418–423
www.elsevier.com/locate/procedia

4th International Conference on Energy and Environment Research, ICEER 2017, 17-20 July
2017, Porto, Portugal

of α-Amylase
Production The and
15th International β-Glucosidase
Symposium from and
on District Heating Aspergillus
Cooling niger
by solid state fermentation method on biomass waste substrates
Assessing the feasibility of using the heat demand-outdoor
from rice husk, bagasse and corn cob
temperature function for a long-term district heat demand forecast
Andi Aliyah, Gandhi Alamsyah, Rizky Ramadhani, Heri Hermansyah*
I. Andrića,b,c*, A. Pinaa, P. Ferrãoa, J. Fournierb., B. Lacarrièrec, O. Le Correc
Department of Chemical Engineering, Faculty of Engineering, University of Indonesia, 16424 Depok, Ind onesia
a
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
b
Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France
Abstract c
Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France

Hydrolysis enzy mes can be produced fro m Aspergillus niger using solid state fermentation method. This research
aimed to understand the fermentation efficiency of A. niger with different variety of the bio mass waste substrates,
Abstract
i.e. rice husk, sugarcane bagasse and corn cob, to produce α-amy lase and β-glucosidase. The optimu m fermentation
time for each substrate was 6 days. The highest activity unit for α-amy lase and β-glucosidase were 81.86 U/ ml and
District
95.02 U/ heating
ml using networks arecorn
substrate commonly addressed inActivity
cob respectively. the literature
unitsaswere
one 73.94
of the U/
most effective
ml and 82.35solutions for decreasing
U/ ml measured the
for dry
greenhouseand
α-amylase gas dry
emissions from the respectively.
β-glucosidase building sector.This
These systems
dried require
enzyme washigh investments
stable which are
for hydrolysis returned
process through
at 30-50 the heat
o C.
sales. Due to the changed climate conditions and building renovation policies, heat demand in the future could decrease,
©prolonging the investment
2017 The Authors. return
Published byperiod.
Elsevier Ltd.
The main scope of this paper
Peer-review under responsibility ofis to assess the feasibility
the scientific committee of using
of thethe4thheat demand –Conference
International outdoor temperature
on Energyfunction for heat demand
and Environment
forecast. The district of Alvalade, located in Lisbon (Portugal), was used as a case study. The district is consisted of 665
Research.
buildings that vary in both construction period and typology. Three weather scenarios (low, medium, high) and three district
renovation scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained heat demand values were
Keywords: Aspergillus niger; biomass; solid state fermentation; α-Amylase; β-Glucosidase
compared with results from a dynamic heat demand model, previously developed and validated by the authors.
The results showed that when only weather change is considered, the margin of error could be acceptable for some applications
1.(the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation
Introduction
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered).
The value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the
Indonesia
decrease in theis number
an agricultural
of heatingcountry
hours ofand in every
22-139h year
during theit heating
produces agro-industrial
season (depending on waste fro m agriculture
the combination sector,
of weather and
forestry,
renovationfarming,
scenarios and industriesOn
considered). . According to BPS
the other hand, [1], Indonesia
function producedfor2600.35
intercept increased tonsperofdecade
7.8-12.7% sugar (depending
cane and 780.1
on the
tons of cane
coupled bagasse.
scenarios). The Bio mass
values derivedcould
suggested frombeagricultural
used to modify wastethe containing carbon for
function parameters sources are stillconsidered,
the scenarios quite large.
and
Carbon
improvecontent in biomass
the accuracy such as estimations.
of heat demand rice husk was 48.9% [2], so it can be used as a substrate in enzyme production
process. Hydrolysis enzyme is enzy me used for breaking glyosidic bonds in polysaccharides into simple sugars.
© 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and
Cooling.
* Corresponding author. Tel. and fax: +62-813-8539-9048; +62-217-863-515.
E-mail address: heri@che.ui.ac.id
Keywords: Heat demand; Forecast; Climate change
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 4th International Conference on Energy and Environment
Research.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 4th International Conference on Energy and Environment
Research.
10.1016/j.egypro.2017.10.269
 Andi Aliyah et al. / Energy Procedia 136 (2017) 418–423 419
H. Hermansyah et al./ Energy Procedia 00 (2017) 000–000

This sugar can be utilized in production of bioethanol. En zy matic hydrolysis could be carried out at mild conditions
(temperature 30-50 °C and pH 5) that does not require a large energy [3]. Hydrolysis enzy mes that we used in the
hydrolysis reaction were α-amylase and β-glucosidase. These α-amylase and β-glucosidase enzymes obtained from
microorganis ms, such as bacteria, fungi and yeast. This study focused on the production of hydrolysis enzymes, α -
amy lase and β-glucosidase from different kind of bio mass substrate using solid-state fermentation method [4-7]. In
this research, agricu ltural bio mass that we used were sugarcane bagasse, corn cobs and rice husk. The experimental
results showed that the fungi Aspergillus niger is capable to produce both enzyme α-amylase and β-glucosidase.

Nomenclature

𝛼𝛼 Alfa
𝛽𝛽 Beta
𝑑𝑑 Diameter of cuvette
∆𝐸𝐸 Absorbance
𝑈𝑈 Enzyme activity
∆𝑡𝑡 Hydrolysis time
𝑉𝑉𝑓𝑓 Final volume including DNS solution, mL
𝑉𝑉𝑠𝑠 Volume of enzyme used, mL
Σ Extinction coefficient

2. Materials and methods

2.1. Materials research

The materials used in the study are PDA (Potato Dextrose Agar), (NH4 )2 SO4 , KH2 PO4 , K2 HPO4 , MgSO4 .7H2 O,
lactose, maltose, phosphate buffer, DNS (Dinitrosalicy lic acid), starch and distilled water. The substrate used were
corn cobs, rice husks, and bagasse. The microorganism used was Aspergillus niger.

2.2. Preparation of fermentation substrate

The substrate were cut into pieces of ≤1 cm and then dried using an oven at 70o C for 24 hours. Bagasse was
pre-treated using autoclave at 121 C for 15 minutes to remove lignin co mpounds. Each fermentation needs 30 g of
substrate and mix it with nutrients such as 0.6 g (NH4 )2 SO4 ; 0.18 g KH2 PO4 ; 0.18 g K2 HPO4 ; 0.03g MgSO4 .7H2 O,
maltose and lactose 1 g and 60 mL H2 O. Then sterilize the substrate for 15 minutes at temperature of 121 C in
autoclave.

2.3. Fermentation

Fermentation was done by SSF method using substrate from corn cobs, rice husks and bagasse, respectively 30 g
for each fermenter. The ratio of nutrients and substrate addition was 1:15 in a 250 mL Erlen meyer. Inoculu m
solution was transferred in the medium 3% (v/v). Incubation was done for 6 days at 30 o C and pH condition at 7.0.

2.4. Enzymes extraction

En zy me extract ion was done by adding 0.1 M phosphate buffer pH 7.0 with the ratio of 1:2 (w/v). The solution
was mixed for 30 min and then filtered using muslin cloth. The extract was centrifuged at 8000 rp m for 20 minutes.
Analysis of the enzy me activ ities were calculated using a decrease in the amount of g lucose in solution by using the
DNS reagent (3,5-dinit rosalicylic acid). Enzy me unit (U) means as the amount of enzyme required to produce 1
μmol of glucose per minute. Enzy me activity was calculated with the value of absorbance at 540 n m. In b rief,
testing was done by adding 0.5 ml o f enzy me sample and added 0.5 ml starch solution 1%. Then, it was incubated
420 Andi Aliyah et al. / Energy Procedia 136 (2017) 418–423
H. Hermansyah et al./ Energy Procedia 00 (2017) 000–000

for 5 min at 30 o C. The addition of 1 ml of DNS reagent stopped the hydrolysis reaction. After that, the solution was
heated at 90 o C for 5 min to react between DNS reagent and the glucose reduction. The measurement of absorbance
was used spectrophotometric UV-Vis with wavelength 540 nm.

2.5. Drying

Drying was used to keep the quality of en zy me remain stable. There were t wo metho ds of drying that was used,
(a) freeze drying perfo rmed in BPPT Serpong and (b) spray drying performed in LIPI Cib inong. Enzy me drying use
freeze dry ing method must added matrix casein 0.05% (w/v) and fo r spray dryer method added matrix skim milk
12% (w/v). Condition operation of spray drying was inlet temeperature at 130 °C and outlet temperature 60-70 °C
with flow rate of hot gas was 1.45 m3 /min solution.

3. Results and discussion

3.1. Effect of fermentation time on enzyme production

Fermentation was done by SSF method with the same amount of substrate in the room temperature. Samples were
collected during fermentation of substrate for measuring the yield of α -amylase and β-glucosidase on day 3, 4, 5,
and 6 (Fig. 1). Based on the data, the highest enzyme act ivity of α-amy lase was measured on the 6th day for every
substrate. In fermentation using rice husk substrate, enzy me activ ity of α-amy lase at 144 hours was 65.95 U/ ml. For
fermentation using corn cob, en zy me activity of α -amy lase at 144 hours was 81.86 U/ ml. For fermentation using
substrate bagasse, enzyme activ ity at 144 hours was 75.35 U/ ml. Fig . 1 shows the effect o f fermentation t ime on
enzyme produced based on enzyme activity.

Fig. 1. Enzyme activity of (a) α-amylase (b) β-glukosidase in various time fermentation.

Based on the data, the highest enzyme acitivity of β-glucosidase was on the sixth day or at 144 hours for every
substrate. In fermentation using rice husk substrate, enzyme activity of β -glucosidase at 144 hours was 31.91 U/ ml.
For fermentation using corn cob, enzyme activ ity of β-glucosidase at 144 hours was 85.01 U/ ml. Fo r fermentation
using substrate bagasse, enzyme activity at 144 hours was 91.67 U/ ml. Aspergillus niger reached stationary phase
between day 4th and 5th on mediu m potato dextrose agar (PDA) [7]. In this study, A. niger grew in agro-industry
biomass, where b io mass contains plurality of substances such lignin, cellu lose and hemicellulose that cannot be
directly processed by this organisms.

3.2. Effect of variation of substrate fermentation of enzyme production

Other purpose of this study was to obtain the best substrate for enzymes production. Bio mass substrates supply a
carbon source for microorganisms producing enzyme wh ich is Aspergillus niger. The selection of the substrate that
 Andi Aliyah et al. / Energy Procedia 136 (2017) 418–423 421
H. Hermansyah et al./ Energy Procedia 00 (2017) 000–000

used in this study based on their potential amount in Indonesia. Based on the data (Fig. 2a), fermentation using rice
husk produced enzyme α-amy lase with enzy me activ ity 65.95 U/ ml. Fermentation using corn cob produced α -
amy lase with en zy me activ ity 81.86 U/ ml, while bagasse produced enzyme α -amy lase with enzy me activity 75.35
U/ml. The highest enzyme activity between three kinds of substrate was produced from corn cob.
Based on the data (Fig. 2b), fermentation using rice husk produced enzy me β -glucosidase with enzy me activ ity
31.91 U/ ml. Fermentation using corn cob produced β-glucosidase with enzy me activ ity 95.02 U/ ml, wh ile bagasse
produced enzyme β-g lucosidase with enzy me activity 91.67 U/ ml. Figure 2 belo w shows the highest enzyme
activity between three kind of substrate.

Fig. 2. Enzyme activity of (a) α-amylase (b) β-glukosidase in various substrates.

When viewed fro m amount of carbon source for each substrate, th ere were not much difference so it did not show
a significant effect. On rice husk, amount of carbon source reached 48.9% [2]. On bagasse, amount of carbon
sources reached 45.5% [8]. On corn cob, carbon sources reached 46.8% [2]. Other factors that affected enzyme
production in solid fermentation system are suitable substrate for microorganisms, substrate pre -treatment process,
particle size, mo isture content, type and size of inoculu m [8]. Moist content and inoculum were fixed variab le in this
study. Moist content for each fermenter was 1:2 (v/v) and amount of inoculum was 3% (v/w).
Rice husk substrate unable to produce enzyme with high activity possibly because rice husk has smaller particle
size than the other two types of substrates. Size of rice husk appro ximately was less than 0.5 cm so inside fermenter
would be compact and caused lack of aeration, wh ich impacted on the growth of Aspergillus niger and affected the
enzy me yield. Whereas in other types of substrates, corn cobs and bagasse, have a larger particle size so that when
the fermentation occurs in erlenmeyer there would have a bit space for aeration.

3.3. Effect of enzyme drying

The drying process will allows decreasing enzy me act ivity or may occur inactivation of the en zy me [9]. Dry ing
methods which commonly used is spray drying and freeze drying method. In drying process, it required
characteristics of the enzy me so the process would not reduce enzy me activ ity. Retention of en zy me activity should
be close to 100% after d rying so the lifetime of the dry enzy me ab le to last longer [10]. Retention of the enzy me can
be calculated by comparing enzyme activity before and after drying process.
In the drying process by using spray drying, inlet and outlet temperatures should be controlled in optimal
condition to obtain high retention of en zy me act ivity. Moreover, outlet temperature was higher than the temperature
of stable enzyme resulted in denaturation of the enzyme [10]. Freeze d rying method produced dry enzyme with
approximately 0.03 g/ ml dry en zy me of liquid extract. Retention of en zy me activ ity by using freeze d rying (Tab le 1)
ranging fro m 84% to 98%. Results of enzyme drying by using spray drying method (Table 2) produced
approximately 0.05 g/ml dry enzyme of liquid extract. Retention of enzyme activity approximately 91-96%.
422 Andi Aliyah et al. / Energy Procedia 136 (2017) 418–423
H. Hermansyah et al./ Energy Procedia 00 (2017) 000–000

T able 1. Retention of dry enzyme activity by using freeze drying method.

T ype of Enzyme T ype of Substrates Retention (%)
α-amylase Rice Husk 84.8%
Corn Cob 87.9%
Bagasse 98.1%

β-glucosidase Rice Husk 86.5%

Corn Cob 95.0%
Bagasse 89.8%

T able 2. Retention of enzyme activity by using spray drying method.

T ype of Enzyme T ype of Substrates Retention (%)
α-amylase Bagasse 96.04
β-glucosidase Bagasse 91,64

3.4. Enzyme stability at various temperature

Hydrolysis reaction generally used for various types of industries such as for sugar industry, textile, food and
other industries. En zy matic hydrolysis process usually occurs at mild operating conditions, lo w temperature up to
100 °C, amb ient pressure, pH 6-8 [11]. Th is research is determining whether the results for both enzymes α-amy lase
and β-glucosidase have stability in hydrolysis reaction with various temperatures. Hydro lysis temperature variations
was at 20, 30, 50 and 70 °C (Fig. 3).
The data shows that α-amylase had highest activity when hydrolysis at temperature of 30 °C and had the lowest
activity at temperature of 20 and 70°C. Based on these data, α-amy lase produced optimally in hydrolysis reaction
was at temperature of 30-50 °C and the enzy me α -amylase had no resistance at low temperature (≤ 20 °C) and
high temperature (≥70 °C). En zy me β-g lucosidase had similar results as α-amy lase. Optimal temperature used for
hydrolysis reaction was at temperature 30-50 °C and en zy me β-g lucosidase had no resistance at low temperature (≤
20 °C) and high temperature (≥ 70°C).

Fig. 3. Enzyme activity of (a) α-amylase (b) β-glukosidase in various temperatures.

4. Conclusion

Based on this study, we concluded that the fermentation time for 6 days still increased in enzy me activity matter
for both enzymes α-amy lase and β-glucosidase by using various types of substrate fermentation. Type of substrate
 Andi Aliyah et al. / Energy Procedia 136 (2017) 418–423 423
H. Hermansyah et al./ Energy Procedia 00 (2017) 000–000

fermentation with highest enzyme act ivity for both enzyme α -amy lase and β-glucosidase is corn cob. Retention of
enzy me activity is about 85-98% after drying process. This enzyme has an optimal temperature fo r hydrolysis
process at temperature 30-50 °C. Production of 100 g dry enzyme, required 600 g of substrate fermentation.

Acknowledgements

The authors acknowledge support from Universitas Indonesia and Ministry of Research, Technology, and Higher
Education Republic of Indonesia in this research.

References

[1] BPS. Produksi Padi, Jagung, dan Kedelai. Jakarta: BPS; 2013.
[2] Wannapeera, Janewit , and Suneerat Pipatmanormai. (2008) “Product yields and characteristics of rice husk, rice straw and corncob during
fast pyrolysis in a drop-tube/fixed-bed reactor.” Songklanakarin J. Sci. Technol 30 (2008):393-404.
[3] Taherzadeh, Mohammad J, and Keikhosro Karimi. (2007) “Enzyme Based Hydrolysis Processes for Bioethanol from Lignocellulasic
Material: A Review.” Bioresources 2.4 (2007): 707-738.
[4] Andi Nur Aliyah, et.al. (2016) “ Solid state fermentation using agro-industrial wastes to produce Aspergillus niger lipase as a biocatalyst
immobilized by an adsorption-crosslinking method for biodiesel synthesis.” International Journal of Technology 7.8 (2016): 1392-1403
[5] Kumar, M. Siddhartha, Chandana Lakshmi, and V. Sridevi. (2013) “Production and optimization of Glucoamylase from wheat bran by
Aspergillus oryzae NCIM 1212 under Solid State Fermentat ion.” International Journal of Application or Innovation in Engineering &
Management (IJAIEM) 2 (2013): 318-323.
[6] Pandey, Ashok. (1999) “ Solid state fermentation for production of industrial enzymes.” Current Science 77 (1999): 149-162.
[7] Khan, Jahir A and Sachin K Yadav. (2011) “Production of Alpha Amylases by Aspergillus niger Using Cheaper Substrates Employing
Solid State Fermentation.” International Journal of Plant, Animal and Environment Science 5 (2011): 100-108.
[8] Arsène, Marie-Ange. (2013) “Treatments of non-wood plant fibres used as reinforcement in composite materials.” Materials Research 16.4
(2013): 903-923.
[9] Pilosof, Ana and Virginia Sanchez. Handbook of Industrial Drying: Drying of Enzymes, 3 rd Edition. Singapore: CRC Press; 2006.
[10] Yoshii, H. (2008) “Effects of protein on retention of ADH enzyme activity encapsulated in trehalose matrices by spray drying.” Journal of
Food Engineering 87 (2008): 34-39.
[11] Kolusheva, T and A Marinova. (2007) “A Study of The Optimal Conditions for Starch Hydrolysis through Thermostable α-amylase.”
Journal of the University of Chemical Technology and Metallurgy (2007): 93-96.