Use of wastewater sludge as a raw material for production of L-lactic acid

Article abstract of DOI:10.1021/es980483v

This study utilizes wastewater sludges to produce L-lactic acid, a precursor of biodegradable plastic. The high concentrations of cellulose contained in the sludge, derived from a paper manufacturing facility, have been found to be convertible to L-lactic acid at a rate as high as 6.91 g/L. To achieve such a high conversion rate, the sludge must be pretreated with cellulase. This pretreatment includes inoculation of the sludge with lactic acid bacteria, strain LA1, after the sludge has been subjected to enzymatic hydrolysis. This study utilizes wastewater sludges to produce L-lactic acid, a precursor of biodegradable plastic. The high concentrations of cellulose contained in the sludge, derived from a paper manufacturing facility, have been found to be convertible to L-lactic acid at a rate as high as 6.91 g/L. To achieve such a high conversion rate, the sludge must be pretreated with cellulase. This pretreatment includes inoculation of the sludge with lactic acid bacteria, strain LA1, after the sludge has been subjected to enzymatic hydrolysis.

Full text of DOI:10.1021/es980483v

Environ. Sci. Technol. 1999, 33, 198-200
(11). The present study is the first to produce lactic acid from
wastewater sludge.
Use of Wastewater Sludge as a
Raw Material for Production of
L-Lactic Acid
Materials and Methods
Sludge and Lactic Acid Bacteria. The present study used
two kinds of sludges derived from wastewater treatment
facilities. One is from the fish-processing industry (sludge A)
and the other from the paper-manufacturing industry (sludge
B). These sludges differed not only in their moisture, organic
matter, and elemental C, H, and N (Table 1) contents but
also in their appearance when dried; i.e., sludge A appeared
sandy while sludge B pulpy.
K I YO H I K O N A K A S A K I , * N A O K I A K A K U R A ,
T O M O H I K O A D A C H I , A N D
T E T S U O A K I YA M A
Department of Materials Science and Chemical Engineering,
Shizuoka University, 3-5-1 Johoku,
Hamamatsu 432-8561, Japan
Lactic acid bacteria were isolated from sludge A. It is well-
known that lactic acid bacteria usually require various kinds
of micronutrient for growth factors; however, it is expected
that the lactic acid bacteria isolated from sludge would grow
and produce lactic acid in the sludge without the need for
additional growth factors, because the bacteria is living in
the sludge in the first place. We selected bacteria that
produced a clear zone on an agar plate cultured at 32 °C for
7 days using the medium for lactic acid bacteria (yeast extract,
5 g; peptone, 5 g; glucose, 20 g; KH2PO4 2 g; distilled water,
1 L; pH ) 6.1) supplemented with CaCO3 (12). Acid-forming
bacteria can be distinguished from others on the agar plate,
and it is easier to identify the lactic acid bacteria from bacteria
consisting only of acid-forming bacteria than from that with
all the bacteria indigenous to the sludge. After cultivation at
32 °C for 7 days, colonies were successively streaked and
purified and then stored on the slants of GYP medium
(glucose, 10 g; peptone, 5 g; yeast extract, 10 g; CH3COONa‚
3H2O, 2 g; MgSO4‚7H2O, 200 mg; MnSO4‚4H2O, 10 mg; FeSO4‚
7H2O, 10 mg; NaCl, 10 mg; Tween 80, 500 mg; distilled water
1 L; pH ) 6.8) at 4 °C. The lactic acid production of each
acid-forming bacterium was then examined in the liquid
medium by determining the concentration of L-lactic acid
with the assay system, TC L-lactic acid (Boehringer Mann-
heim, GmbH), that utilizes an enzymatic reaction of NAD-
linked L-lactic dehydrogenase with L-lactic acid for producing
NADH. The concentration of L-lactic acid was determined
by measuring the absorbance of NADH at 340 nm. The
bacterium that most effectively produced L-lactic acid, strain
LA1, was used for conducting further experiments. The strain
LA1 grew at even a high concentration of glucose, 180 g/ L,
and the productivity of L-lactic acid at the early stage of the
fermentation was 0.24 g/ L/ h, which is similar to the rates of
production reported previously (10, 11, 13, 14).
This study utilizes wastewater sludges to produce L-lactic
acid, a precursor of biodegradable plastic. The high
concentrations of cellulose contained in the sludge, derived
from a paper manufacturing facility, have been found to
be convertible to L-lactic acid at a rate as high as 6.91 g/L.
To achieve such a high conversion rate, the sludge must
be pretreated with cellulase. This pretreatment includes
inoculation of the sludge with lactic acid bacteria, strain LA1,
after the sludge has been subjected to enzymatic
hydrolysis.
Introduction
Sludge is disposed in large quantities from wastewater
treatment facilities and continues to be one of the most
troublesome waste materials to deal with. The conversion of
waste biomass into chemical feedstocks may reduce our
dependence on petroleum-derived feedstocks while adding
a value to hitherto value-less waste materials. The conversion
of the cellulose in sludge to lactic acid is a step toward
achieving environmental sustainability, while finding a new
value in waste sludge. The present paper describes the
hydrolysis of cellulose from sludge and the subsequent
conversion to L-lactic acid, a precursor of biodegradable
plastic.
A variety of biodegradable plastics have recently been
developed (1). However, their high production costs are an
important issue the industry must tackle to promote the use
of biodegradable plastics. Thus, it is urgently needed to find
some means to bring down the production costs. Peimin has
estimated the production costs of L-lactic acid and has
concluded that the cost of raw materials is ca. 40% of the
total manufacturing costs (2). Therefore, achieving a low
production cost for L-lactic acid is dependent primarily on
what we choose for raw materials. If wastewater sludge can
be efficiently used as a raw material for the production of
polylactic acid, then a considerable reduction in costs would
be possible.
How to best utilize the cellulosic materials has been a
topic of active current research. Many studies have been
published (e.g., refs 3-7) regarding the production of ethanol,
i.e., biofuel from cellulosic material. Some studies are
concerned with production of lactic acid from cellulosic
materials that is usable in various industries (8-10). Recently,
a study has been done to use lactic acid, produced from
waste paper, as a raw material for a biodegradable plastic
The LA1 strains was inoculated into sludge A and sludge
B without pretreatment, and the production of L-lactic acid
in each sludge was examined.
Analysis of Glucose and Polysaccharide Concentration
in the Sludge. The sludges were dried at 105 °C for 2 days
and then powdered by a mincer (<0.2 mm in diameter). The
concentrations of glucose and polysacchardes that could be
converted to glucose by hydrolysis were measured for each
sludge. Glucose was extracted from the sludge by boiling it
for 2.5 h in distilled water. The ratio of sludge to distilled
water was 1:9 on a weight basis. In the analysis of the
polysaccharide concentrations, the polysaccharides were
hydrated by the method of Inoko et al., a method that was
developed to measure the cellulose and hemicellulose
concentrations in compost (15). Hydrolysis was carried out
by adding 5 mL of an 80% H2SO4 solution to 1.25 g of dried
sludge at temperatures 12-15 °C for 2.5 h. The mixture was
then diluted with 175 mL of distilled water and boiled for 5
h.
* Corresponding author phone/ fax: 81-53-478-1172; e-mail:
tcknaka@eng.shizuoka.ac.jp.
9
1 9 8 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 1, 1999
10.1021/es980483v CCC: $18.00
1998 Am erican Chem ical Society
Published on Web 12/01/1998

After the extraction of glucose or the hydrolysis of the
polysaccharides, the concentration of glucose was deter-
mined by the Glucose C-II test WAKO (Wako Pure Chemical
Industry, Ltd.) assay system that utilizes an enzymatic
reaction of mutarotase and glucose oxidase with glucose for
producing H2O2. The H2O2 then reacts with 4-aminoantipy-
rine and phenol by the action of peroxidase, producing red
quinone pigment. The concentration of glucose was deter-
mined by measuring the absorbance of the pigment at 505
nm.
To confirm the validity of the measurement methods
employed in determining the concentrations of glucose and
polysaccharides, we added known quantities of standard
substances, glucose and cellulose, to the dried sludge and
then measured the concentrations again. It was confirmed
that the concentrations of glucose and polysaccharides can
be accurately determined by the methods adopted in this
experiment.
TABLE 1. Characteristics of Two Kinds of Sludges
sludge A
sludge B
m oisture content (%)
organic m atter contenta (%)
elem ent contenta
carbon (%)
78.5
79.1
49.9
70.7
38.7
33.1
0.51
4.86
0
nitrogen (%)
9.30
hydrogen (%)
5.66
0
0.87
glucose (%)
polysaccharidesb (%)
33.3
a
b
Dry weight basis. The concentration of polysaccharides was
expressed in term s of glucose generated by hydrolysis (glucose
equivalent concentration).
Enzym atic Hydrolysis of the Sludge. Enzymatic hydroly-
sis of sludge B was carried out by mixing 30 g of dried sludge
with a 500 mL acetate buffer solution (pH ) 4.5) containing
0.1% cellulase and keeping it at 45 °C for 7 days. The cellulase
used was Meicelase CEPB-5081 which was derived from
Trichoderma viride and had an activity of 6180 U/ mg (1000
U corresponds to an activity of 1 g of filter paper being
completely degraded in 1 min) at 37 °C. It was ascertained
that the cellulase exhibits its maximum activity at around 45
°C with pH ) 4.5. The transient glucose concentration, with
the progress of hydrolysis, was determined by the method
described above. Aseptic conditions were m aintained
throughout the experiment.
Ferm entation of Sludge Hydrolysate. In order to activate
the microbial activity, the LA1 strain of lactic acid bacteria
was precultured twice and successively (16): the LA1 strain
was first inoculated into the centrifuging tube containing a
30 mL GYP liquid medium. After incubation of the culture
at 30 °C for 2 days, the cell of LA1 was centrifuged at 15 000
rpm for 10 min, and the supernatant was exchanged for a 30
mL fresh liquid medium and then incubated again at 30 °C
for 2 days.
After the second preculture, the cell of LA1 was centrifuged
at 15 000 rpm for 10 min, and the supernatant was exchanged
for 30 mL of enzymatic hydrolysate of sludge B, which was
prepared as described above but incubated only 2 days and
then autoclaved at 121 °C for 15 min to deactivate the
cellulase. The cell density of the LA1 strain at the start of the
hydrolysate fermentation was measured by dilution plating.
During the hydrolysate fermentation experiment, the
concentrations of glucose and L-lactic acid were measured
daily by the method described above. The cell density of the
LA1 strain in the sample was also measured by the dilution
plating method.
FIGURE 1. The course of glucose concentrations and the conversion
of polysaccharides during the enzymatic hydrolysis of sludge B.
The conversion of polysaccharides is defined as the ratio of the
glucose concentration at a given time to the glucose concentration
generated as a result of complete hydrolysis, i.e., acid hydrolysis.
the sludge shows a low N content. In view of this, it may be
assumed that a large quantity of cellulose flows out into the
wastewater during the paper-manufacturing process, which
then condenses into sludge, resulting in a high cellulose
content in the sludge.
Next, we performed an enzymatic hydrolysis of sludge B.
The conversion of polysaccharides was assessed. Here, the
conversion was defined as the ratio of the glucose concen-
tration at a given time to the glucose concentration generated
after acid hydrolysis has been completed. In the present
experiment, 30 g of dried sludge (10 g glucose-equivalent
polysaccharides) was hydrated in a 500 mL solution; therefore
20 g/ L glucose was produced with complete hydrolysis of
the polysaccharides. The final conversion of polysaccharides
at 98 h is ca. 50%, which implies that 10 g/ L of glucose is
produced (Figure 1). The polysaccharide conversion did not
exceed 50% probably because of the inhibition of cellulase
due to glucose, i.e., through product inhibition, or the
deactivation of cellulase during the hydrolysis, and/ or the
presence of polysaccharides other than cellulose in sludge
B.
Results and Discussion
It was first confirmed that lactic acid was not produced when
the LA1 strain was inoculated into the two sludges without
pretreatment. The concentrations of glucose and polysac-
charides that could be converted to glucose by acid hydrolysis
were then determined (Table 1). No glucose was detected in
either of the two sludges, whereas the quantity of polysac-
charides that could be converted to glucose by acid hydrolysis
was greater in sludge B than in sludge A. The polysaccharide
concentration expressed as a glucose-equivalent concentra-
tion in sludge B was as high as ca. 33% (Table 1). This high
concentration of polysaccharides in sludge B appears to be
unique for a wastewater sludge. We have not yet analyzed
the constituent polysaccharides in the sludge B. We assume,
however, that these polysaccharides include cellulose, since
the sludge was obtained from a wastewater treatment facility
of the paper manufacturing industry. As shown in Table 1,
Finally, we attempted to produce L-lactic acid from the
hydrolysate of sludge B. The cell density of the LA1 strain at
the start of fermentation was 9.2 log10 CFU/ mL. The final
concentrations of L-lactic acid and glucose after 5 days of
fermentation were 6.91 and 0.48 g/ L, respectively, showing
that L-lactic acid can be produced from the wastewater sludge
by combining hydrolysis and fermentation processes (Figure
2). The LA1 strain produced L-lactic acid in the sludge
hydrolysate without supplementation of any growth factors.
The production yield of L-lactic acid from glucose was found
to be ca. 0.9 g-lactic acid/ g-glucose, which was quite high,
indicating that a large portion of consumed glucose was
9
VOL. 33, NO. 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 1 9 9

and China (2). The costs of using sludge as a raw material
for the production ofL-lactic acid have not yet been estimated.
Extra costs will inevitably be incurred, when compared with
the case of using corn starch as a raw material, for the
hydrolysis of the cellulose in the sludge. On the other hand,
corn starch must be acquired at a cost, while the sludge may
be acquired free of charge. Furthermore the cost of sludge
treatment could be charged on a revenue basis. It would be
possible, therefore, to produce L-lactic acid from sludge at
$1 per kilogram or less, although the cost may vary depending
on the costs of hydrolysis and sludge acquisition. To reduce
the overall costs for producing L-lactic acid from wastewater
sludge, it is important to optimize the hydrolysis of the sludge
as well as the fermentation process of the hydrolysate. Further
study is needed to achieve a higher efficiency in hydrolyzing
cellulose and attaining a higher concentration of L-lactic acid
in the sludge hydrolysate.
Acknowledgments
This research was supported partly by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science
and Culture of Japan. The authors also express their thanks
to Meiji Seika Kaisya Ltd. for a supply of Meicelase.
Literature Cited
FIGURE 2. The course of concentrations of glucose and L-lactic
acid, and of the cell density of strain LA1 during sludge hydrolysate
fermentation (O: L-lactic acid, b: glucose, [: LA1).
(1) Biodegradable Plastic and Polymers; Doi, Y., Fukuda, K., Eds.;
Elsevier: Amsterdam, London, New York, Tokyo, 1994.
(2) Peimin, Y. Ph.D. Dissertation, The United Graduate School of
Agricultural Science, Shizuoka University, 1997.
(3) Lynd, L. R.; Cushman, J. H.; Nichols, R. J.; Wyman, C. E. Science
1991, 251, 1318-1323.
converted to L-lactic acid with high selectivity. The cell density
gradually decreases as the fermentation progresses, finally
reaching about 7.9 log10 CFU/ mL (Figure 2). Hence it can
be considered that the pH decrease associated with lactic
acid production have inhibited the growth of the LA1 strain
itself. In future experiments, we plan to control the pH during
the fermentation of sludge hydrolysate by adding CaCO3 or
through the use of a pH stat instrument.
L-Lactic acid can be made from sludge B, which contains
a high concentration of cellulose, provided that the hydrolysis
and fermentation processes are combined. Sludge B is
discharged at a rate of 35 000 t/ year, which corresponds to
ca. 1.4% of the total amount of sludge from the paper-
manufacturing industry in Japan. By assuming that the
polysaccharides contained in the sludge from the paper
manufacturing industry can be completely hydrated into
glucose and then completely converted to L-lactic acid, the
production rate of polyactic acid amounts to 420 000 t/ year,
which corresponds to ca. 3% of the total plastic materials
production rate in Japan.
(4) Philippidis, G. P. In Handbook on Bioethanol: Production and
Utilization; Wyman, C. E., Ed.; Taylor & Francis: Washington,
D.C. 1996; pp 253-285.
(5) Lee, J. J. Biotechnol. 1997, 56, 1-24.
(6) Wymann, C. E. Bioresour. Technol. 1994, 50, 3-15.
(7) Wymann, C. E.; Goodmann, B. J. Appl. Biochem. Biotechnol.
1993, 39/40, 41-59.
(8) Abe, S.; Takagi, M. Biotechnol. Bioeng. 1985, 27, 389-397.
(9) Venkatesh, K. V.; Wanket, P. C.; Okos, M. R. AIChE Symp. Ser.
1994, 90, 80-87.
(10) Xavier, S.; Lonsane, B. K. Appl. Microbiol. Biotechnol. 1994, 41,
291-295.
(11) Schmidt, S.; Padukone, N. J. Ind. Microbiol. Biotechnol. 1997,
18, 10-14.
(12) Sasaki, H. Memoirs of the Faculty of Agriculture, Hokkaido
University (in Japanese); 1971; Vol. 8, pp 188-247.
(13) Kurosawa, H. Biotech. Bioeng. 1988, 31, 183-187.
(14) Peimin, Y.; Nishina, N.; Kosakai, Y.; Yahiro, K.; Park, Y.; Okabe,
M. J. Ferment. Bioeng. 1997, 84, 249-253.
(15) Inoko, A.; Sugahara, K.; Harada, Y. Soil Sci. Plant Nutri. 1979,
25, 225-234.
(16) Okada, S.; Otuka, B. Seibutsu-Kogaku Kaishi (in Japanese) 1996,
74, 305-308.
Peimin has stated that it is most effective to reduce the
cost of raw materials in order to minimize the total L-lactic
acid manufacturing cost and that it may be possible to
produce L-lactic acid with a production cost of less than $1
per kilogram from corn starch through the use of the newly
developed air-lift bioreactor (14) in countries like the U.S.A.
Received for review May 12, 1998. Revised manuscript re-
ceived October 27, 1998. Accepted October 29, 1998.
ES980483V
9
2 0 0 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 1, 1999