Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29
DOI 10.1186/s40643-016-0106-8

Open Access

RESEARCH

Lactic acid production from paper
sludge by SSF with thermotolerant Rhizopus sp.
Maki Takano and Kazuhiro Hoshino*

Abstract
Background: Rhizopus fungi is suitable for the production of lactic acid, which is the backbone material of poly‑lactic acid used as green plastic from lignocellulosic biomass, since it can grow and ferment in simple medium withvarious carbon sources such as starch and cellulose. Although paper sludge (PS) contains a lot of cellulosic fibers
and in general was incinerated for volume reduction and heat recovery, other efficient utilizations have hardly beendeveloped. In effective production of lactic acid from PS, the research of the extraction of cellulosic fiber from raw PS
to obtain effectively fermentable sugars by cellulase and the selection of lactic acid microorganism are necessary. In
this study, the PS pretreatment method with NaOH and HCl and the optimization of cellulase reagent were achieved,
and also a desirable thermotolerant Rhizopus was selected. Finally, the production of lactic acid from the treated PS at
40 °C by simultaneous saccharification and fermentation (SSF) with the strain and an optimized cellulase cocktail wasinvestigated.
Results: Rhizopus oryzae NBRC 5384 was selected for thermotolerant lactic acid production from Rhizopus librarybecause of its heat tolerance up to 40 °C and high lactic acid production of 80 g/L. The strain can ferment to lactic
acid from hexose, pentose, sugar alcohol, disaccharide and starch. The soaking of raw PS in NaOH and HCl was able
to reduce effectively inorganic materials and other reagents for repulping, and the content of Al and Ca per PS dry
matter was mainly decreased from 32.9 and 30.8 to 14.1 and 1.66 %, respectively. SSF of the treated PS of 50 g/L with
optimized cellulase cocktail and 5384 at 40 °C resulted in lactic acid production of 9.33 g/L for 96 h.
Conclusion: The thermotolerant Rhizopus fungus was found based on its high performance in lactic acid produc‑tion at high temperature from not only glucose, but also other various carbon sources including polysaccharides andthe secretion of amylases and cellulases. The treatment of raw PS by NaOH and subsequent HCl was able to remove alarge amount of inorganic materials with decrease of hydrophobicity. In SSF of the treated PS with the strain and the
optimized cellulase cocktail, lactic acid was able to be produced. However, the increase of initial PS concentration in
SSF led to the decrease of the yield with ethanol production, because of limited aeration due to increase of density.
An appropriate oxygen supply to the strain is necessary to improve lactic acid production.
Keywords: Lactic acid, Paper sludge, Thermotolerant, Rhizopus oryzae, SSF
Background
Petrochemical production has caused worries amid concern about depletion of fossil fuel. Additionally, disposal
treatment of a lot of the waste plastic is also a major consideration. As one of the sustainable green plastic materials, polylactic acid synthesized from lactic acid has
been used increasingly as an alternative to petrochemical*Correspondence: khoshino@eng.u‑toyama.ac.jp
Graduate School of Science and Engineering, University of Toyama,3190 Gofuku, Toyama 930‑8555, Japan

plastics. It can be discarded into the soil because of its
biodegradability; the matter may be decomposed by
microorganisms and used as carbon sources for plants
and microorganisms. Lactic acid and its derivatives arealso important chemicals that have many applications
in food, chemical and pharmaceutical products (Passos et al. 1994; Datta et al. 1995; Lunt 1998; Grade et al.
2002). While it is produced by chemical synthesis, thismanufacturing method results in a racemic mixture of dand l-lactic acids. As enantiopure lactic acid is required© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.

Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

to produce polylactic acid which is stable structurally
and does not contain impurities, lactic acid production
by bioconversion has attracted attention because of its
economical procedure compared with chemical synthesis. Moreover, the biological production method of lactic
acid is valid because of the possibility of using lignocellulosic materials as feedstock.
Rhizopus fungi belonging to Zygomycota are wellknown microbes of enantiopure l-lactic acid from glucose and other carbon sources. The fungi are also able
to ferment starch to lactic acid (Ping Huang et al. 2005;
Yin et al. 1997; Zhou et al. 1999). Furthermore, several researches have been reported about the fungus, bywhich cellulolytic and xylanolytic enzymes were secreted(Bakir et al. 2001; Murashima et al. 2002). The saprophytic fungus has high potential for lactic acid production from various abundant cellulosic biomasses, suchas agricultural (rice straw, wheat straw), forestry (wood)and industrial waste (old paper, paper sludge) (Lynd et al.
1991, 1999; Lynd et al. 2001; Wyman 2003; Gao et al.
2008; Marques et al. 2008; Hama et al. 2015). Rhizopus
fungi can produce l-lactic acid from simple media withnot only several monosaccharides, but also starch andlignocelluloses with lower production cost than that byother lactic acid bacteria though many kinds of microorganisms such as Lactobacillus, Lactococcus and Enterococcus (Woiciechowski et al. 1999; Thongchul and Yang
2003; Wang et al. 2010). Additionally, Rhizopus can produce three major end products of l-lactic acid, fumaricacid and ethanol, unlike homofermentative lactic acidbacteria. These products are affected by culture condition, and sufficient aeration induces efficient lactic acid
(Cui et al. 1998; Skory et al. 1998).
Paper sludge (PS) is a solid residue arising from paper
manufacture and the generated amount is more than 3.5
million ton per year in Japan, according to statistic dataon industrial waste from paper manufacturing by theJapan Paper Association. Since it is mainly incinerated
to reduce the volume and regenerate thermal energy,
the use of a lot of fossil fuel for the discard treatment
has been viewed with suspicion, because PS has highmoisture content up to 50 % and incineration discharges
a large amount of CO2. Also, the utilization of PS ash
derived from the incineration is limited and is used asfilling materials for cement, asphalt and rubber, or as amaterial for manufacturing cement (Ishimoto et al. 2000).
Though a small part of PS is used as a soil conditioner
and a fertilizer in agriculture, a large part of ash is disposed of in sanitary landfills (Henry 1991; Tripepi et al.
1996). Therefore, new technology of effective utilizationof PS should be developed for not only the productionof sustainable materials as alternative to petrochemicals,

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but also the reduction of paper waste. PS is a promisingmaterial as one of a lignocellulosic feedstock for lactic
acid production from the content of a large amount ofshort cellulosic fibers already treated by delignificationof paper making. While PS has already been transmuted
to cellulosic fiber in the pulping process by digestion
with alkali and steam, general pretreatment of lignocellulosic biomasses needs high energy to delignify andexpose the cellulose fiber. However, the PS contains alarge amount of paper filler materials, such as CaCO3,
kaolin (Al2Si2O5(OH)4), talc (Mg3Si4O10(OH)2) and otherinorganic materials, and it is possible to be an inhibitor of enzymatic reaction and metabolism of microorganism for the bioconversion of PS. The fillers and theother additives in PS envelop the cellulose fibers andobstruct the access of microbes and enzymes to the cellulose substrate (Nikolov et al. 2000). Cellulase hydrolysis
is especially inhibited by CaCO3, because the pH of thisreaction suspension (pH 6–8) is relatively higher than the
optimum pH of cellulases. (Kang et al. 2010). Although
CaCO3 is essential for pH control for lactic acid production to suppress pH decrease by the acid, removal of ash
in PS leads to effective hydrolysis of PS and lactic acidproduction. CaCO3 can be removed by acid dissolution
or CO2 treatment (Kang et al. 2011).
In this study, lactic acid production by an appropriate
Rhizopus fungus from PS that removed the inorganic
materials was investigated using simultaneous saccharification and fermentation (SSF) method. In SSF, the enzymatic hydrolysis step of cellulose and the fermentative
step by microorganisms are typically combined and thecultivation is operated on a one-batch system (Abe andTakagi 1991; Yanez et al. 2003). Unfortunately, appropriate temperatures for the enzymes and microbes arein general very different. As optimum temperature ofcellulases are generally 40–60 °C and the growth temperature of the common lactic acid bacteria is around
30 °C, SSF must be operated at the growth temperature which causes the cellulase activities to decrease. To
achieve effective SSF, thermotolerant Rhizopus strain was
selected. Several strains can grow and produce lactic acidup to 40 °C (Kitpreechavanich et al. 2008). Conversion
of PS to lactic acid by SSF was studied with thermotolerant Bacillus; however, it was not able to secrete cellulases (Marques et al. 2008; Naresh and Zhiliang 2009).
Since Rhizopus sp. is able to secrete cellulases and hemicellulases, SSF will be able to perform with addition ofrelatively low-dose cellulase economically. Therefore, theselection of thermotolerant Rhizopus strain, removal of
CaCO3 from raw PS and lactic acid production from thetreated PS by SSF with the strain and cellulase cocktail
were investigated.

Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

Methods
Materials

To evaluate lactic acid production, 55 kinds of Rhizopus
sp. was obtained from NBRC (NITE Biological Resource
Center, Chiba, Japan). These strains were maintained on
Difco™ potato dextrose agar (PDA) slants at 4 °C.
The paper sludge (PS) used in this study was supplied
from the final waste sludge discharge spout of deinked
pulp manufacturing process in Chuetsu Pulp & Paper
Co., Ltd. (Toyama, Japan). The moisture content of PS wasaround 50 % by squeezing of the filler-pressed machine.
It was sealed in plastic Ziploc bags and stored at −30 °C
until experimental use. PS was analyzed after oven drying
at 50 °C to constant weight. The PS components of cellulose, hemicellulose, lignin, acid-soluble lignin and ash
were assayed by Klason lignin method (Lopez et al. 2010).
Cellulases in this study used 15 kinds of commercial reagents as follows: Meicelase (Meiji Seika Pharma,
Japan), Cellulase T “Amano” 4, Cellulase A “Amano” 3,
Pectinase G “Amano”, Hemicellulase “Amano” 90(Amano
Enzyme, Japan), Cellulase Y-NG, Cellulase “Onozuka”
3S, Cellulase “Onozuka” R-10 (Yakult Pharmaceutical Industry, Japan), Sumizyme AC, Sumizyme X, Sumizyme SNX (Shin-Nihon Kagakukogyo, Japan), Cellulase
Trichoderma viride, Novozyme 188 (Sigma-Aldrich Co.,
St Louis, MO), Accellerase® (DuPont, DE, USA) and
Vecelex (Godo-Shusei, Japan).
Removal of inorganic materials from raw PS by alkali
and acid solution

Raw PS was suspended in 1 M NaOH solution with
200 g/L consistency and soaked overnight. After thePS fiber was separated from the alkaline liquid, it was
resuspended in appropriate HCl solution of the required
concentration for neutralization of both residual NaON
and CaCO3 contained in PS and soaked overnight. The
PS was separated from the acid solution and thoroughly
washed with water. The PS treated by NaOH and HCl
was oven dried at 50 °C, completely dried and then powdered finely in a mill.
Selection and determination of optimum cellulase cocktail

The treated PS of 50 g/L was hydrolyzed by each 15
kinds of commercial cellulase at the protein concentration of 1 g/L in 100 mL bottles containing 25 mL acetic
acid buffer (pH 5.0). These were shaken in a rotary shaker
at 120 rpm at 40 °C for 48 h and then hydrolysates wereseparated by filtration (Filter paper No.131, Advantec Co.,Tokyo, Japan) and analyzed by HPLC. The cellulase reagents that were able to produce a lot of fermentable sugar(glucose and xylose) from the PS were selected, and theirblending ratio was determined by the design of experiment method using Design Expert 8 (Stat-Ease, Inc. MN).

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Cultivation of Rhizopus fungi

Fungi were precultured on PDA plate at 40 °C until they
covered the overall surface. The mycelia on a plate weremilled using a blender (Milser LM-Plus, Osaka chemical Co., Osaka, Japan) in 100 mL saline per plate and it
was used as inoculums. The composition of the culture
medium was 120 g/L glucose, 3 g/L (NH4)2SO4, 0.3 g/L
KH2PO4, 0.25 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O,
and 50 g/L CaCO3. All reagents used were purchasedfrom Wako Pure Chemical Industries, Ltd (Osaka, Japan).
The medium of 25 mL was put in a 100 ml medium bottle and then sterilized at 121 °C for 15 min. After getting
cold, the cap of the medium bottle was exchanged with asilicone plug (Type C: Asone Co. Tokyo, Japan). Cultivation of fungi was started by inoculation of 1 mL hyphasuspension into the medium and then incubated in arotary shaker (120 rpm) at 40 °C. After the cultivation,
5 ml of 5 M HCl was added to each bottle for dissolution of CaCO3, and the broth was separated by filtration.
Residual sugar and lactic acid in the culture were analyzed by HPLC.
Enzymatic hydrolysis and simultaneous saccharificationand fermentation (SSF) of the treated PS

For lactic acid production from treated PS, 50 or 100 g/Lof the treated PS was used as substrate instead of glucose.
Medium components and the PS in 500 mL baffle flasks
were sterilized at 121 °C for 15 min. Enzymatic hydrolysis was started by the addition of filter-sterilized cellulase cocktail, which consisted of three enzyme reagentsat a calculated rate and protein with a concentration of
3.5 g protein/L (containing endo-β-glucanase, endo-βxylanase, cellobiohydrolase, β-glucosidase, β-xylosidase
and FPase whose activities were 10.4, 30.4, 0.214, 2.44,
0.0429 and 0.473 U/mL, respectively), into the medium
and then shaking at 130 rpm for 144 h at 40 °C. SSF wasstarted by the addition of 8 mL of homogenized mycelium suspension and the cellulase cocktail, and then they
were cultured at the same condition of hydrolysis. Hydrolysates and broths sampled from cultures were analyzed
by HPLC for determination of sugars and lactic acid.
Analytical method

In the case of Klason lignin method, glucose was measured by Glucose C II-test Wako (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) and total sugar was measured by the phenol–sulfuric acid method. Elemental
composition in the ash of PS was determined by X-ray
fluorescence analysis (XRF) using wave length-dispersive
X-ray spectroscopy (PW2404, Spectris Co., Ltd., Tokyo,
Japan). The amount of glucose, xylose, and lactic acidfrom each cultivation was determined by HPLC (LC10ADvp, Shimadzu Co., Kyoto, Japan) equipped with aTakano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

Page 4 of 10

refractive index detector (RID-10A, Shimadzu Co.) andICSep WA-1 Wine Analysis column (Transgenomic, NE,
USA) operated at 40 °C with 1.25 mM sulfuric acid as
mobile phase at flow rate of 0.6 mL/min.

Result and discussion
Removal of inorganic materials from PS

Since raw PS is the final residue of recycled paper manufacturing, PS containing many components with the
exception of cellulose and hemicelluloses fibers such as
CaCO3 from loading materials, reagents and surfactantsby the deinking and the repulping. These materials havethe possibility of becoming inhibitors of hydrolysis of
enzymes and fermentation of microorganism, especially,a large amount of CaCO3 covering the cellulose fibermight obstruct the attack of enzymes and microbes. To
remove these inhibitors for the effective hydrolysis of PS
and subsequent fermentation, their removal process by
dissolution with alkali and acid solution was investigated.
First, raw PS was soaked in 1 N HCl solution to dissolve
CaCO3 in PS. The treatment resulted in the decrease of
the component ratio of inorganic materials from 41.9 to
28.7 % with increase in the ratio of cellulose and othersugars of 14.0 and 8.4 %, respectively, as shown in Table 1.
While partial inorganic materials were removed from raw
PS by the HCl treatment, the treated PS became strong
water-shedding fibers because of the exposure of thehydrophobic surface of the fiber by uncovering of CaCO3.
Next, raw PS was soaked in 1 N NaOH before the HCltreatment in an effort to reduce the hydrophobicity with
dissolution of reagents and surfactants used on repulping
adsorbed on PS by the alkali solution. The NaOH–HCl
treatment resulted in the augmentation of hydrophilicity
with removal of 24.5 % inorganic materials and increaseof cellulose and other sugar contents to 51.3 and 22.4 %,Table 1 Components ratio in raw and each treated PS
Content (%)

Raw

HCl

NaOH–HCl

Cellulose

22.4

36.3

51.3

Other sugars

10.1

18.6

22.4

Inorganic materials

41.9

28.7

17.4

Others

25.5

16.3

8.88

respectively. The elemental components ratio in PS ash
by X-ray fluorescence analysis is shown in Table 2. The
reduced inorganic materials were mainly Ca, Al, and Fe
elements in PS and the decrease ratios were 29.1, 18.9,
and 1.84 %, respectively. From these results, it was shownthat the NaOH–HCl treatment method was excellent forthe disposal of CaCO3 from raw PS.
Selection of thermotolerant lactic acid‑producing strain

To select the best strain that was able to grow at 40 °C
and concurrently to produce effectively lactic acid among
55 species of Rhizopus library, they were first cultured at
40 °C on a PDA plate. Forty species in the library could
well grow under the environment. Next, they were cultured in liquid medium under the aerobic condition at
40 °C, and then the lactic acid produced for 144 h was
determined. Figure 1 shows that lactic acid production
by the fungi selected as the thermotolerant strain. It wasrecognized that 22 kinds of species were able to produce
it. Since R. oryzae NBRC 5384, 5413 and 31,005 was produced more than 50 g/L lactic acid from 120 g/L glucose,
three strains was re-cultured and the detailed investigation of the products was performed. As a result, threestrains produced mainly lactic acid with a small amountof fumaric acid as shown in Table 3. Maximum lactic acidconcentrations were produced were at about 66–74 g/L
for 120 h. Though R. oryzae NBRC 31,005 strain wasreported to preferentially produce fumaric acid (47.8 g/L)
with a small amount of lactic acid (9.7 g/L) at 30 °C (Yinet al. 1997), it was able to produce conversely high lactic
acid with fumaric acid of 0.22 g/L at 40 °C in this study.
Yet on thermotolerance, it is noteworthy that only R.
oryzae NBRC 5384 was able to grow up to 50 °C. Therefore, the R. oryzae NBRC 5384 strain was selected as athermotolerant fungus for the production of lactic acidbecause of the highest heat tolerance.
Lactic acid production by R. oryzae NBRC 5384
by difference of incubation temperatures

R. oryzae NBRC 5384 was cultured with 120 g/L glucose
as a carbon source at 28, 36, 40, and 42 °C for 144 h andthe result is shown in Fig. 2. In all cultures, glucose wasalmost consumed and lactic acid was produced with theassimilation. It was found that the strain was also able toTable 2 Elemental components ratio of ash in raw and treated PS
Ratio
of ashElemental components (%)a
Al

Ca

Si

Mg

Fe

Na

S

P

Ti

Cl

K

Mn

Zn

Cu

Raw

1

32.9

30.8

16.4

5.77

3.55

3.24

2.95

1.99

0.643

0.490

0.426

0.412

0.244

0.114

Treated

0.537

14.1

1.66

24.3

6.07

1.71

2.53

0.743

0.905

0.840

0.216

0.401

0.07590.06840.115

aBased on the content of inorganic material (ash) in raw PS

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Fig. 1 Comparison of lactic acid production from glucose by various Rhizopus strains. Lactic acid concentration produced from 120 g/L glucose byeach Rhizopus strain cultivated for 144 h at 40 °C

Table 3 Comparison of lactic acid and fumaric acid produced by three strains of Rhizopus
StrainLacticacid (g/L)

Fumaric
acid (g/L)

NBRC 5384

69.8

0.164

NBRC 5413

65.9

0.0581NBRC 31005

73.9

0.167

All cultures were carried out for 120 h at 40 °C

Fig. 2 Effect of temperature on lactic acid production from glucose
by R. oryzae NBRC 5384. Cultivation was performed at the initial
glucose concentration of 120 g/L at the temperature of 28, 36, 40,
and 42 °C

produce lactic acid at 42 °C. Lactic acid was produced up
to about 80 g/L in 120 h except for the culture at 42 °C,
although the rates were different at each temperature.
The rates of lactic acid production increased with temperature rising until 36 °C and decreased at the temperature up to 42 °C. The productivities were 0.894 (72 h),

1.11 (72 h), 0.692 (120 h) and 0.462 g/L/h (144 h), respectively. From these results, it is found that 5384 strain isapplicable to lactic acid production at high temperatureof about 40 °C; then it was decided that the following cultures is mainly carried out at 40 °C to maintain stable andeffective fermentation.
Characterization of lactic acid production from several
carbohydrates by R. oryzae NBRC 5384

Lactic acid productions by R. oryzae NBRC 5384 from
15 kinds of carbohydrates (hexoses, pentoses, sugar
alcohol, disaccharides and polysaccharides) as carbon
source were carried out at 40 °C to characterize the lactic acid fermentation ability of the strain. The culture was
performed with 50 g/L of each carbon source for 120 h
and the produced lactic acid and the yields are shown in
Table 4. The strain was able to convert efficiently hexoses such as glucose, galactose, mannose and fructose tolactic acid, and in these cultures the yields of lactic acidbased on the initial carbon sources were about 0.62–0.65.
The strain was also able to ferment to lactic acid fromnot only hexoses, but also slightly from pentoses such as
xylose and arabinose. Moreover, although common lactic
acid microorganism cannot use sugar alcohols for lactic
acid production, the strain was able to ferment sorbitol
slightly. Though maltose and cellobiose were converted
to lactic acid by the stain, lactose was not fermented.
These results means that the strain could produce αand β-glucosidases though β-fructofuranosidase could
not be produced. Since direct fermentation of soluble
starch to lactic acid was able to be carried out, the secretion of α-amylase and/or glucoamylase by the strain was
also suggested. α-cellulose was not able to ferment under
the culture condition. It is thought that the hydrolysis
of α-Cellulose by cellulases such as endo-β-gluconase,Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

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Table 4 Ability of R. oryzae NBRC 5384 to produce lactic
acid using various carbon sources
Lactic acid (g/L)

Yield (g/g)aGlucose

34.2

0.648

Galactose

32.7

0.654

Mannose

31.0

0.620

Fructose

32.9

0.658

Xylose

5.23

0.105

Arabinose

0.13

0.0026

Sucrose0.243

0.00486

Maltose

36.5

0.730

Cellobiose

6.55

0.131

Lactose00

Glycerol00

Sorbitol

8.31

0.166

Mannitol00

Soluble starch

6.80

0.136

α-Cellulose00

All cultures were carried out for 120 h at 40 °C
aBased on the initial concentration of each sugar (50 g/L)

cellobiohydrolase, β-glucosidase et al. was not enoughfor the lactic acid production although the strain was
secreted the slightly several cellulases (data is not shown).
To produce lactic acid from cellulosic biomass with thefungus, it is necessary to add cellulases into the culture.
Especially, it is necessary to execute an SSF combiningof cellulase and thermotolerant fungus for the lactic acidproduction from treated PS, including short cellulose
fiber that has a stronger structure than α-cellulose by SSF.

reagents was performed by Design of Experiment (DOE)
method with Design Expert 8.0. for effective hydrolysis of treated PS. The ratio calculated using DOE was
O:A = 0.157:0.843. Additionally, 0.5 g protein/L Pectinase G (P) was used as a supplement of β-xylosidase
activity for xylose production from hemicellulose intreated PS, because the activities of two selected reagentswere weak. From these results, a desirable cellulase cocktail was constructed at the ratio of O:A:P = 0.47:2.53:0.50(total concentration was 3.5 g-protein/L).
For confirmation of hydrolysis ability against treated PSof the cocktail, substantiative hydrolysis was performed
with the cellulose cocktail at 28 and 40 °C. Figure 3
shows the fermentable sugars (glucose and xylose) produced from 100 g/L treated PS (4.73 FPU/g-PS) at 28
and 40 °C. The hydrolysis rate was increased with risingtemperature, the maximum concentrations of glucose
were 32.1 and 38.3 g/L for 72 h, respectively, and the glucose yield at 40 °C was 0.747 based on cellulose content
in the treated PS. However, xylose production was only
increased slightly by higher temperature. Though theexistence of CaCO3 in the reaction medium was caused
at pH 6–8 and it induced inhibition of cellulase activity,hydrolysis efficiency of cellulose was achieved up to 70 %and improved obviously with rising temperature. Thiscan lead to increase of fermentation efficiency by SSF.
Figure 4 shows the lactic acid production from PS
treated by SSF at 40 °C, combining a thermotolerantstrain with the cellulase cocktail. Glucose and xylose
were produced rapidly at the initiation of culture andthen consumed until the cultivation time of 24 h, andsubsequently not detected. At 24 h, lactic acid began to

Lactic acid production from PS treated by SSF
with thermotolerant fungus

Moreover, the hydrolysis rate of the cellulosic fiber might
be greatly controlled by the compounding ratio of these
enzymatic activities depending on the type of substrate.
Although the commercial cellulase reagent, which hasvarious cellulase activities, was optimized to hydrolyze
effectively targeting cellulosic materials, the cellulase
reagent for hydrolysis of PS is not available and effective hydrolysis is difficult by only a commercial reagent.
Therefore, the blending of several commercial reagentsis a promising strategy to obtain fermentable sugars
from treated PS. First, the hydrolysis reaction of treated
PS by 15 kinds of commercial reagents was carried out
for selection at each concentration of 1 g protein/Land 50 g PS/L for 24 h to compare the initial hydrolysis
rates. Among all the reagents, Cellulase Onozuka 3S (O)and Accellelase (A) were produced from most fermentable sugars (total amount of glucose and xylose) fromthe PS. Next, the optimization of commercial cellulase

Fig. 3 Glucose and xylose production from the PS treated with
optimum cellulase cocktail. Enzymatic hydrolysis of the PS at theinitial concentration was 100 g/L with cellulase cocktail containing
3.5 g-protein/L and the reaction was performed at 28 and 40 °C

Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

Page 7 of 10

Fig. 4 Lactic acid production by SSF of the treated PS with 5384
strain and cellulase cocktail. The initial concentration of the PS was
50 g/L and the cultivation was performed at 40 °C with cellulase
cocktail containing 9.46 FPU/g-PS

Fig. 5 Lactic acid production by SSF of the treated PS with 5384
strain and cellulase cocktail. The initial concentration of the PS was
100 g/L and the cultivation was performed at 40 °C with cellulase
cocktail containing 4.73 FPU/g-PS

produce suddenly and its concentration reached 9.33 g/L
in 96 h. The yield of lactic acid based on the sugar produced from the treated PS was 0.475. The reason for thedelay in the beginning of lactic acid production is that in
the initial stage of culture, the fungus was grew using theenergy obtained by sugar metabolism under aerobic condition and then the metabolism shifted to the lactic acidproduction side by accumulating the intracellular energy
unlike in the case of the mechanism of a general lactic
acid bacteria under anaerobic condition.
Meanwhile, in this SSF, neither ethanol nor fumaric acid, which were common by-products of lactic acidfermentation by Rhizopus sp., was detected. Althoughlactic acid was produced from PS treated by the SSF, its
productivity of 0.244 g/L/h was less than that of the culture in the glucose substrate (0.667 g/L/h). Therefore,
improvement of the lactic acid production by increasing the substrate concentration was attempted. Figure 5
shows the SSF treated with 100 g/L PS at 40 °C. During
culture, lactic acid and ethanol were simultaneously produced from 24 h and then lactic acid (7.56 g/L, at 48 h)and ethanol (5.25 g/L, at 36 h) reached the maximum values. The yield of lactic acid at that point was 0.272 thatwas less than that with 50 g/L treated PS. The lower yield
is attributed to decrease of mass transfer in culture andfluidity by the increase of initial substrate concentration,
because the treated PS was mainly composed of shortlight fibers. Moreover, judging by the fact that ethanol
was produced at an initial culture, the environment inculture might induce suffocation through the SSF. That is,lactic acid production by the 5384 strain requires abundant oxygen and the depletion leads to shift to a pathway
from pyruvate to ethanol production and not to lactic

acid production. From these results, at the start of SSF,
a large amount of cell which accumulates enough energy
intracellularly will produce lactic acid immediately, andthe improvement of suffocation may be able to improvethe delayed lactic acid. Compared to other researches
on lactic acid production from several feedstocks, theyields and the productivities were low in this researchon PS. For example, SSF of starch such as cassava, ricebran and wheat by Lactobacillus sp. resulted in the yields
of 0.66, 0.28 and 0.89 and the productivities of 0.41, 0.78
and 1.74 g/L/h, respectively (Chookietwattana 2014; Tanaka et al. 2006; Hetényi et al. 2011). In other researchesof lactic acid from alfalfa fiber and cassava bagasse by
the same species, the yields were 0.35 and 0.96 and the
productivities were 0.75 and 1.40 g/L/h, respectively(Sreenath et al. 2001; John et al. 2006). While these feedstocks are pure materials obtained from agriculture, PS
used in this study is industrial waste and contains a lotof harmful components such as inorganic materials andvarious reagents for repulping, which inhibit hydrolases
and microorganism. Since pH in culture is rendered
faintly alkaline with CaCO3 which is the loading material
for pulping and suppressor for pH decrease by the lactic
acid produced, the activities of cellulases that have optimum pH at mild acid were depressed and the production of fermentable sugars was limited. Then lactic acidproduction by the fungus was delayed. Furthermore, theaeration of culture is important for enhancement of lactic acid production, because the 5384 strain used in thisstudy responds sensitively against dissolved oxygen andswitches ethanol production to lactic acid production
by adequate oxygen supply. To improve the yield andTakano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

productivity, investigation of the PS treatment method
and cultivation condition should be carried out in detail.
If the SSF process is accomplished with high efficiency, as
the PS is a complex material and difficult to ferment thanthe other general cellulosic biomasses, it must be able toapply to other biomasses.
On the bioconversion from lignocellulosic biomass,
which is applied not only to lactic acid but also to theother fermentation products, SSF is a more valid process
than separate hydrolysis and fermentation (SHF) because
the process can be carried out in one tank batch, thoughhigher yields of fermentation products are achieved by
SHF than SSF since it is possible to start high initial fermentable-sugars by the concentration of the hydrolysate(Hetényi et al. 2011). The reason why SSF progressed the
production of lactic acid effectively is that it can avoid
product inhibition against hydrolases by fermentablesugars, as the lactic acid fermentation microorganisms
consume sugars formed by hydrolases at once. Although
the SSF is commonly adopted for fermentation, it has aproblem about difference of appropriate temperatures
between cellulases and fermenting-microorganisms, andtherefore the process is in general forced to execute at the
low temperature agreed with an optimum temperaturefor fermenting microbes despite cellulases cannot keep
maximum potential. To overcome the temperature gap,
the application of thermotolerant fermenting-microorganisms for high-temperature SSF is effectual. It will be able
to achieve the production of a large amount of fermentable sugars immediately by high cellulase activity and thesubsequent fermentation. Enhancement of cellulases activity by increasing the temperature can lead to a reductionof cellulase usage in SSF and therefore the reduction in
production cost will be accomplished. Moreover, though
for low-temperature SSF the biomass has to be sterilized,
since lignocellulosic biomasses includes a large amountof environmental microbes such as bacteria and fungi,
the high-temperature SSF has the advantage that the contamination from biomass can be avoided easily. Therefore,
there are many possibilities for the use of thermotolerant
zymogenic microorganisms as biocatalyst for bioconversion of lignocellulosic feedstock to fermentation products.

Conclusion
With the slow depletion of fossil resource such as crude
petroleum, the utilization of untapped natural resources
has been received worldwide attention. The economical
production of green plastic from lignocellulosic materials
by bioconversion has recently been of interest in terms ofcarbon-neutral. Polylactic acid is one of the green plastic and lactic acid that is a raw material widely used in
the field of food, chemical and pharmaceuticals, etc.
In this research, paper sludge (PS) excreted as a waste

Page 8 of 10

product from the paper-making plant was focused onas an unused cellulosic material, because in the circumstances the treatment is in general carried out by incineration with a large amount of fossil fuel and therefore
carbon dioxide has been generated. In addition, the PS
ash is industrial waste and stable supply is more possible
all the year round.
The production of lactic acid from PS containing a greatamount of cellulose material was investigated using simultaneous saccharification and fermentation (SSF) with a
new-found thermotolerant Rhizopus fungus and optimized cellulase cocktail. The screening of the fungus thatcan not only grow, but also ferment glucose to lactic acidat 40 °C was performed among the Rhizopus library in ourlaboratory, and some fungi belonging to R. oryzae and R.
microsporus were able to grow at more than 40 °C andproduce lactic acid. Taking the tolerance to high temperature and the ability of lactic acid production into consideration, R. oryzae NBRC 5384 was selected as the fungus
for the production of lactic acid by SSF of PS. The strain
was able to produce lactic acid from glucose in high yieldunder the aerobic condition at 40 °C. Moreover, various
sugars such as hexose, pentose, disaccharide and starchwere directly bioconverted to lactic acid by the fungus.
These results suggested that the strain can secrete α- andβ-glucosidases, α-amylase and glucoamylase.
Since in common SSF, the hydrolysis process was a
rate-limiting step, and the improvement of its hydrolysis
rate was the most important task for the effective production of lactic acid. To remove the unnecessary inclusion such as inorganic materials and deinked reagentsused for paper manufacturing which inhibit cellulases
activity and/or fermentation by thermotolerant fungus,
the pretreatment of raw PS was investigated. PS treatment of soaking in NaOH and subsequently HCl wasvery efficient in the exposure of cellulosic fiber from PS,
though its hydrophilicity was increased. Ca from CaCO3
in the loading material and Al from Al2Si2O5(OH)4 in
kaolin were mainly decreased by the treatment. Removalof inhibitors against enzymatic reaction by cellulases andfermentation by the fungus were efficiently executed withrecovery of the cellulosic fiber in PS.
The optimization of cellulase reagent was performed by
the DOE method based on the total amount of fermentablesugar (glucose plus xylose) produced from the treated PS
with 14 kinds of commercial cellulase reagents. The total
amount of fermentable-sugars formed from the treated PS
by using the cocktail at 40 °C was vastly superior to thatat 28 °C. Moreover, lactic acid was able to be produced by
SSF of the treated PS at 40 °C with a new thermotolerant
fungus, Rhizopus oryzae NBRC 5483, combined with thedesirable cellulase cocktail. In the culture condition withhigh substrate concentration, the stagnation of oxygen

Takano and Hoshino ﻿Bioresour. Bioprocess. (2016) 3:29

supply to the strain might be caused the change of metabolic pathway. In particular, limitation of oxygen supply
led to not only lactic acid, but also ethanol production.
As it now stands, although the amount of lactic acidproduced from the treated PS by the SSF at 40 °C was low
and the bioconversion has a problem relating to oxygen
supply, the SSF might be one of promising methods forthe utilization of PS as an unused cellulosic waste without CO2 emission, pushing ahead with the research as
quickly as possible. The SSF process from PS will be able
to achieve the production of various biomaterials such as
not only lactic acid, but also other organic acids or ethanol in the near future.
Authors’ contributions
TM carried out all PS treatments, enzymatic reactions, cultivations and analy‑
ses. TM has been involved in drafting the manuscript. HK has been involved
in revising it critically for important intellectual content. HK has given final
approval of the version to be published. Both authors read and approved thefinal manuscript.
Acknowledgements
This research was supported by A-STEP program (Industrial needs responsetype) of the Japan Science and Technology Agency (JST).
This study was presented at 12th Asian Congress on Biotechnology(ACB2015). Topic: Bioenergy and Biorefinery.
Competing interests
The authors declare that they have no competing interests.
Received: 4 January 2016 Accepted: 23 May 2016

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