JOURNAl.

OFBIOSCmNCEANDBIOENGINEERING
Vol. 91, No. 4, 339-343. 2001

Conversion of Chitinous Wastes to Hydrogen Gas by Clostridium

paraputrificum M-21
DWIERRA EVVYERNIE, t KENJI MORIMOTO, 2 SHUICHI KARITA, I TETSUYA KIMURA, I
K A Z U O S A K K A , 1 AND KUNIO OHMIYA 1.
Faculty o f Bioresources, Mie University, 1515 Kamihama, Tsu 514-8507 a and Aichi Science and Technology
Foundation, Naka, Nagoya 460-0002, 2 Japan
Received 16 October 2000/Accepted 2 December 2000

The chitinolytic bacterium Clostridium paraputriflcum strain M-21 produced 2.2 and 1.5 mol hydrogen gas
from 1 mol N-acetyl-v-glucosamine (GlcNAc) and ball-milled chitin equivalent to I tool of GicNAc, respec-
tively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster
shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol It2 from 1.5 g of the latter.
Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment.
Waste from the starch industry was also converted to hydrogen. When (7. paraputriflcum M-21 was cultivated
on ball-milled chitin and baH-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or
Chill were detected as the major chitinase species in the supernatant of the cultures, suggesting that they play
a critical role in the degradation of chitinous materials.
[Key words: hydrogen production, Clostridium paraputrificum, chitin, chitinase, shrimp shell, waste]

Since energy consumption in the world has increased
tremendously, we are facing a shortage of energy in the
near future. Hydrogen offers huge potential benefits in
terms of reduced emissions of pollutants and greenhouse
gases and it is a premium-quality energy carrier due to
the generation of energy at a level 3 times higher than
that generated by methane. Hydrogen can be produced
from feedstocks including natural gas, coal, or nuclear
sources (1, 2). However, these sources have problems
related to shortage and/or safety. To resolve these pro-
blems, biomass which is photosynthesized by solar energy
is now regarded as a sustainable future energy source,
since it can be metabolized into hydrogen by hydrogen-
generating microorganisms. Hydrogen-generating micro-
organisms have been identified among photosynthetic
bacteria (3-5), cellulolytic anaerobes (6-10) and chitinoly-
tic bacteria (11).
One abundant biomass is marine biomass such as
shrimp shell which consists mostly of chitin. Chitin is a
homopolymer of N-acetyl-D-glucosamine (GIcNAc) linked
by t3-1,4-glycosidic bonds, and functions a role as a struc-
tural polysaccharide in many marine organisms, insects,
fungi and algae (11-14). Chitin can be degraded by mi-
crobes via hydrolysis of the glycosidic bond by chitinases.
Chitinases are a group of enzymes capable of degrading
chitin to low-molecular-weight products and the key
enzymes which initiate the solubilization of chitin (15, 16).
Among chitinolytic bacteria, Clostridium is known to
ferment chitin to produce hydrogen (11). In a previous
paper, we reported that Clostridium paraputrificum M-
21 isolated from barnyard soil in Mie University campus
produced high quantities of hydrogen from GIcNAc, glu-
cose and cellobiose, and was considered a potent hydro-
gen producer (17).
In the present paper, we describe the conversion of
ball-milled chitin and raw marine and agricultural wastes
to hydrogen by C. paraputrificum M-21 under controlled
* Corresponding author, e-mail: ohrniya@bio.mie-u.ac.jp
phone: +81-(0)59-231-9622 fax: +81-(0)59-231-9684
339
medium pH during cultivation. We also deal with the
production of chitinases in the culture supernatant of
strain M-21 grown on chitinous materials.
MATERIALS AND METHODS
Bacterium C. paraputrificum M-21 (17), a chitino-
lyric and hydrogen-generating anaerobic bacterium, was
used.
Substrates We used GlcNAc, chitin (Nacalai Tesque,
Kyoto), and wastes such as shrimp shell, lobster shell and
corn fiber as substrates in the cultivation of C. parapu-
trificum M-21. Chitin was ball-milled in water for 3 d.
The shrimp and lobster shells were obtained from a
restaurant in Tsu city. The former were thin shells with
a creamy colour and the latter were thick and tough with
an orange colour. These shells were used as carbon
sources with or without acid and alkali pretreatment.
Shrimp shells were pretreated according to the modified
method of Stanley et al. (18): they were ball-milled in
1 N HC1 solution for 5 d and then washed with 1 N
KOH. The resulting precipitate was recovered by cen-
trifugation for 30min at 9000g and resuspended in
water, and the pH of the suspension was adjusted to 7.0
with 1 N KOH. Lobster shells were pretreated according
to the modified method by Wang et al. (16). The shells
were well ground using a mortar and pestle, demineral-
ized by ball-milling in 2 N HCI for 2 d, deproteinized in
2 N NaOH for 30 rain and washed with water. The result-
ing sample was finally dried at 65C and kept at 4C
before use. Corn fiber was obtained from a company as
it is a by-product (Shikishima Starch MFG Co. Ltd.,
Suzuka, Mie). This was very thin and light weight, con-
sisting of 7-10//oo moisture, 11-13% protein, 20-22%
starch, 20--22% fiber, and 33--42% unidentified materi-
als.
Cultivation of C. paraputrificum M-21 Strain M-
21 was cultivated anaerobically in modified GS medium
(17) in a jar fermentor under strictly anaerobic condi-
tions at 45C for the given period at the given pH. The
340 EVVYERNIEET AL. J. BIOSCLBIOEr~6.,

working volume of the medium in a 1-1 jar fermentor TABLE 1. Hydrogenevolution from the culture in a jar fermentor
(type MDL, B.E. Marubishi Lab., Tokyo) was 500ml containing GIcNAcor ball-milled chitin
with an agitation speed of 250 rpm. We used one of the GlcNAc Ball-milled chitina
above substrates at a concentration of 1% as the main Medium
carbon source. The medium pH was maintained at the Yield Yield
pH Hydrogen (molH2/mol Hydrogen
given pH by the automatic addition of 10M NH4OH (l/l medium) GlcNAc) (I/I medium) (molH2/mol
GIcNAc)
during cultivation. Antifoam was added to the medium 5.5 1.81 2.3 -- --
to control foaming. 5.8 1.87 2.4 1.17 1.2
Nitrogen gas was gently blown into the autoclaved 6.0 1.72 2.2 1.50 1.5
medium in the jar fermentor to ensure anaerobic condi- 6.3 1.44 1.7 -- --
tions before inoculation. 6.5 1.08 1.3 1.43 1.4
S D S - P A G E and zymogram analysis To precipitate
a It was assumed that 1g of chitin consisted of 4.5 mmol GIcNAc
proteins, acetone was added to the supernatants of the residues.
C. paraputrificum cultures to give a final concentration --, Not tested.
of 40% and the supernatants were incubated at - 2 0 C
for 1 h. The proteins were pelleted by centrifugation at was the highest at pH 5.8 with a yield of hydrogen of
15,000g for 15min, dried and redissolved in the approximately 2.4 tool H2/mol GlcNAc. Previously, we
sample buffer for SDS-PAGE. After heat treatment at found that C. paraputrificum M-21 produced hydrogen
100C for 3 min, the sample was subjected to SDS- with a yield of 1.9mol H2/mol GIcNAc in medium
PAGE (8%) (19). Chitinase activity was detected on the without pH control. These observations suggest, that
SDS-PAGE gel containing glycol chitin by zymogram maintaining the culture pH at a constant level is im-
analysis (20). portant for enhancing the hydrogen productivity of C.
Western blotting analysis The protein samples paraputrificum M-21. When insoluble ball-milled chitin
from the culture supernatants were separated by SDS- was used as the substrate, the yield of hydrogen was the
PAGE and transferred electrophoreticaUy onto a nitrocel- highest at pH 6.0, i.e., 1.5 mol H2/mol GIcNAc equiva-
lulose membrane using an electroblotting apparatus, lent. In this study, we assumed that 1 g of ball-milled
Sartoblot II (Sartorius, Goettingen, Germany) (21). Im- chitin contained 4.5 mmol GlcNAc. The optimum pH for
munoblotting was carried out using an anti-ChiA or the growth of C. paraputrificum M-21, 6.0, was consis-
anti-ChiB antibody as described elsewhere (20, 22). tent with that for the optimum pH of enzyme activity of
Enzyme and protein assays Chitinase activity was the major chitinases ChiA (unpublished results) and
measured by the modified method of Schales (23) with ChiB (21).
colloidal chitin as a substrate. One unit of chitinase In the subsequent experiments in which insoluble
activity was defined as the amount of enzyme which materials were used as carbon sources, the pH of the
produced 1/~mol of reducing sugar as an N-acetylgluco- medium was maintained at 6.0 during cultivation. Figure
samine standard per min. Protein concentrations were 1 shows the time course of the cultivation of C. para-
determined by the method of Lowry (24) with bovine serum putrificum M-21 in medium containing 1~0 ball-milled
albumin (Sigma, USA) as a standard. chitin as a carbon source. The hydrogen evolution rate
Analytical methods During cultivation in the jar was estimated after 2 h of cultivation to be 1.1 mmol/l
fermentor (batch culture system), gas production was medium/h and it increased exponentially from 4 h (1.3
measured using a wet gas meter (W-NK Da-0.5A, Sina- mmol/l medium/h) to 10 h (8.1 mmol/l medium/h), reach-
gawa Co., Tokyo) and this was used to calculate the rate ing the highest rate after 12h (9.3 mmol/l medium/
of gas evolved. To determine the extent of degradation h), and then decreasing as a result of the consumption
of chitin, shrimp shell or other wastes during cultiva- of the substrate. In this cultivation, the total amount of
tion, the residual insoluble materials in the culture broth hydrogen evolved was 1.5 1/1 medium or 67.0 mmol H2/I
were collected and dried at 105C. The extent of degra- medium, with almost complete consumption of the sub-
dation of chitin was calculated based on the dry weight strate (9.9g//), and the final hydrogen evolution yield
of residual substrates after the cell weight was accounted was 1.5 mol H2/mol GIcNAc equivalent.
for using a standard curve of the correlation between the
cell dry weight and hydrogen production. The hydrogen 80 12
content was measured using Gas Chromatograph GC-
323 online with VStation Chromatography Data System
vers. 1.6 (Compaq Deskpro, GL Sciences Inc., 1998) and
the amount of hydrogen evolved was calculated based
~.= 40 6 E
on hydrogen standard curves.

RESULTS AND DISCUSSION

l- 1.. 0 .~
Hydrogen evolution from ball-milled chitin at different ~ - 0 . . . . o
pHs In a previous study, we found that the initial ~ ~ 0 4 s lz t6 zo
pH of the culture medium strongly affected the total Cultivation time (h)
amount of gas evolved from GlcNAc by C. paraputrificum
FIG. I. Timecourse of cultivation of C. paraputrificum M-21 on
M-21 (17). Therefore, the hydrogen productivity from ball-milled chitin in batch culture with the pH adjusted to 6.0, at 45C
GIcNAc or ball-milled chitin was investigated under con- and 250 rpm. The cultivationwas carried out as described in Materials
ditions where the pH was controlled by the addition of and Methods. Symbols: ", hydrogenevolved(mmol/t); O, hydrogen
NH+OH during cultivation (Table I). When the substrate evolution rate (mmol/I/h); A, residual amount of ball-milled chitin
was GlcNAc, a soluble saccharide, hydrogen evolution (g/t).
Vot. 91, 2001
12
~2 20
8-1~
4 q~'r"
~ O-
t6 20 0
Culttvatton time (h)
25 12
.B
~ 10~
~_~ 015
'~'~ 10
t9 ~ ~
~ , 20 0
16
Cultivation time (h)
FIG. 2. Time course of cultivation of C. paraputr~icum M-21 on
raw shrimp shell (A), and raw lobster shell (B) with the pH adjusted
to 6.0, at 45C and 250 rpm. The cultivations were carried out as
described in Materials and Methods. Symbols: , hydrogen evolved
(mmol//); O, hydrogen evolution rate (mmol/l/h); A, residual
amount of substrate (g//).
H y d r o g e n evolution from raw chitinous wastes
capacity o f C. paraputro'icum M-21 to degrade natural
substrates was tested by cultivating it on raw shrimp
and lobster shells (chitinuous wastes) as the carbon
sources using j a r fermentor. Chemical analysis showed
that the chitin content o f shrimp and lobster shells was
29% and 24%, respectively. As shown in Fig. 2A, the
hydrogen evolution rate for raw shrimp shell was lower
than that for ball-milled chitin, e.g., although the highest
rate was o b t a i n e d after 4 h o f cultivation ( 2 . 7 m m o l
HE/I m e d i u m / h ) , this value was a b o u t one third o f the
m a x i m u m value obtained for ball-milled chitin (9.3 m m o l
H2/I m e d i u m / h ) . H y d r o g e n evolution ceased after 12 h
o f cultivation with shrimp shell as the carbon source.
F o r raw lobster shell, the hydrogen evolution rate
reached a plateau (1.1 H2/I m e d i u m / h ) after 2 h o f cul-
tivation which was m a i n t a i n e d for a b o u t 4 h (Fig. 2B).
H y d r o g e n evolution from the lobster shells ceased after
10 h o f cultivation. The totai a m o u n t o f hydrogen evolved
was 11.4 m m o l / ! m e d i u m (395 ml/! medium) as a result
o f the c o n s u m p t i o n o f 2 . 6 g o f shrimp shell// medium
and 7.8 m m o l / ! m e d i u m ( 2 7 0 m l / / medium) as a result
o f the c o n s u m p t i o n o f 1.5 g o f lobster shell/! medium.
The yields were calculated to be 1.0 and 1.2 tool H2/mol
G l c N A c equivalent for shrimp a n d lobster shells, respec-
tively, when it was assumed that only chitin in the sub-
strates was consumed.
H y d r o g e n e v o l u t i o n f r o m pretreated chitinous wastes
Figure 3 shows the effects o f acid and alkali pretreat-
ments o f the chitinous wastes on hydrogen evolution
in j a r fermentors. Acid and alkali pretreatment o f the
shrimp and lobster shells almost completely removed
proteins and minerals, i.e., the pretreated materials ap-
peared to be relatively pure chitin. As shown in Fig. 3A,
CONVERSION OF WASTES TO HYDROGEN GAS 341
25 t . 12 4
s ,0. /
o" ; " s n 20 0 e~
Cultivation time (h)
25 12 4
4 B
~ 20 to "~
~ ~". O,
,( 4 8 12 16 20 24 28
gl.
Cultivation time (h)
FIG. 3. Time course of cultivation of C. paraputrificum M-21 on
pretreated shrimp shell (A), and lobster shell (B) with the pH adjusted
to 6.0, at 45C and 250 rpm. The cultivations were carried out as
described in Materials and Methods. Symbols: m, hydrogen evolved
(mmol//); o , hydrogen evolution rate (mmol/l/h); A, residual
amount of substrate (g//).
The the highest rate was obtained for pretreated shrimp shell
after 10 h o f cultivation (4.4 m m o l H2/l m e d i u m / h ) ; this
value was two fold higher than the m a x i m u m value
obtained for raw shrimp shell, but still two fold lower than
that obtained for bail-milled chitin. The total a m o u n t o f
hydrogen evolved was 2 1 . 0 m m o l H2/l medium or 469
ml/l m e d i u m as a result o f the c o n s u m p t i o n o f 4.1 g o f
s u b s t r a t e / ! medium. P r e t r e a t m e n t o f the lobster shells
also resulted in a 2-fold increase in the total a m o u n t o f
hydrogen evolved by C. paraputrificum M-21, but the
hydrogen evolution rate was not significantly affected
(Fig. 3B). The total a m o u n t o f hydrogen evolved from
the pretreated lobster shells was 1 4 . 9 m m o l / l m e d i u m
(334ml/1 medium) as a result o f the solubilization o f
2.5 g o f s u b s t r a t e / / medium. The yields were 1.2 and
1.3 mol H2/mol G l c N A c equivalent for the pretreated
shrimp and lobster shells, respectively. The pretreatment
increased the total a m o u n t o f hydrogen evolved two fold
although this bacterium could not completely hydrolyze
the pretreated materials.
Detection o f ehitinases in the culture supernatants of
C. paraputriflcum M-21 grown on chitinous substrates
Chitinases are responsible for the hydrolysis o f chitin.
Therefore, we investigated the p r o d u c t i o n o f chitinases
by C. paraputrificum M-21 grown on bail-milled chitin
and ball-milled shrimp shell. As shown in Fig. 4A-a,
when C. paraputrificum M-21 was cultivated with bail-
milled chitin as a carbon source, m a n y proteins in the
culture supernatant were detected on the S D S - P A G E gel
by Coomassie brilliant blue staining. Z y m o g r a m analysis
indicated that some o f them had chitinase activity. In
a previous paper (20), we reported that chitinases C h i A
(89,000Da) and ChiB (87,000Da) were the m a j o r
chitinase species o f C. paraputrificum M-21 and that
342 EVVYERNIE ET AL. J. BiOSCl. BioEu~.,

12 14 16
A 12 14 16 M 12 14 16
213 kDa
-94 kDa 94 kDa 120 kDa
-67 kDa -67 kDa
76 kDa
-43 kDa 43 kDa 47 kDa

b .

12 14 16 18 M 12 14 16 18

-94 kDa 12 14 16 18
-94 kDa -213 kDa
-67 kDa -120 kDa
B -67 kDa
- 76 kDa
-43 kDa
-43 kDa -47 kDa

a b c
FIG. 4. Results of SDS-PAGE, zymogram, and Western blot analysis of proteins in the culture supernatant of C. paraputrificum M-21 grown
on ball-milled chitin (A) and ball-milledshrimp shell treated with acid and alkali (B) from batch culture at a controlled pH of 6.0. (a) Gel stained
with Coomassie brilliant blue; (b) chitinase activity detected on a gel containing 1% glycol chitin; (c) ChiA and ChiB proteins detected in the
culture supernatant by Western blot analysis with ChiA and ChiB antisera. Lanes: M, size markers; 12-18, the samples cultivated for 12-18 h.

these enzymes cannot be distinguished by either SDS-
P A G E or Western blot analysis using antisera against
C h i A and ChiB because o f their similarity in size and
immunological properties. Western blot analysis showed
that a protein b a n d at the 87,000-89,000 D a position was
immunoreactive with the a n t i - C h i A antiserum (Fig. 4A-
c), confirming that C h i A a n d / o r ChiB is the m a j o r
chitinase o f this bacterium. Chitinases other than C h i A
and ChiB detected by z y m o g r a m analysis did not react
with the a n t i - C h i A antiserum, suggesting that these
enzymes are not proteolysis products o f C h i A or ChiB.
W h e n ball-milled shrimp shell was used as a carbon
source for strain M-21, the S D S - P A G E gel profile o f pro-
teins in the culture supernatant was quite simple, i.e., a
protein b a n d o f a p p r o x i m a t e l y 88,000 Da protein b a n d
was detected as the m a j o r protein and some faint bands
were also observed (Fig. 4B-a). The m a j o r protein(s)
showed chitinase activity (Fig. 4B-b) and reacted with
the a n t i - C h i A antiserum, strongly suggesting that C h i A
a n d / o r ChiB was the m a j o r chitinase o f strain M-21.
Some chitinases o f 43,000 to 60,000 Da were detected by
z y m o g r a m analysis although these proteins were not
clearly identified by Coomassie brilliant blue staining.
The zymographic profile o f the proteins shown in Fig.
4B-b was similar to that shown in Fig. 4A-b, suggesting
that the same chitinases were p r o d u c e d by C. para-
putrificum M-21 regardless o f the substrates used. A
similar zymographic profile was obtained f r o m the cul-
ture supernatant o f strain M-21 grown on lobster shells
(data not shown).
This bacterium p r o d u c e d some proteins without
chitinase activity when grown on bail-milled chitin. It is
likely that some c o m p o n e n t s in the commercial chitin
induced the p r o d u c t i o n o f proteins other than chitinases.
Therefore, when this bacterium was cultivated on ball-
milled shrimp shells, C h i A a n d / o r ChiB was p r o d u c e d
at higher levels than other proteins, suggesting that some
components in the raw shells might induce the produc-
tion o f both chitinases but not o f other chitinases or pro-
teins.
Hydrogen evolution from starch industry waste
A l t h o u g h C. paraputrificum M-21 was charaterized as a
chitinolytic bacterium, we tested the capacity o f this
bacterium to convert starch industry waste to hydrogen.
Using a j a r fermentor, strain M-21 p r o d u c e d 13 m m o l
o f hydrogen per liter o f m e d i u m f r o m corn fiber in a 6-h
cultivation with the c o n s u m p t i o n o f a p p r o x i m a t e l y 2.1
g/l o f the starch waste (Table 2). The yield calculated for
corn fiber was 1.1 mol HE/mOl glucose consumed, when
it was assumed that the substrate consumed was starch.
The values for glucose a n d cellobiose were 1 and 1.4 tool
H 2 / m o l glucose consumed, respectively. A l t h o u g h the
extent o f c o n s u m p t i o n o f the starch waste was low,
t h e yield o f hydrogen was c o m p a r a b l e to those obtained

TABLE. 2. Hydrogen evolution from starch waste containing glucose polymers

Substrates (1%) Type of cultivation Cultivation period Consumption of Total hydrogen production Yield
(h) suhstrates
(g//) (retool H2/I medium) (mol H2/mol glucose consumed)
Corn fiber jar fermentor 6 2.1 13 1.1
Cellobiose jar fermentor 6 10.0 80 1.4
Glucose jar fermentor 6 10.0 63 1.1
VOL. 91, 2001
for glucose a n d cellobiose.
In this study, we f o u n d that strain M-21 is c a p a b l e o f
solubilizing a n d f e r m e n t i n g n o t o n l y c h i t i n o u s m a t e r i a l
but also starch waste, i n c l u d i n g cellulosic materials, to
p r o d u c e h y d r o g e n . T h e s e findings are consistent with
o u r p r e v i o u s o b s e r v a t i o n that C. paraputrificum M-21
can solubilize starch a n d esculin h a v i n g /3-glycosidic
linkages (17). T o o u r k n o w l e d g e , this is the first r e p o r t
o n h y d r o g e n - p r o d u c i n g b a c t e r i a t h a t can c o n v e r t b o t h
c h i t i n o u s a n d starchy m a t e r i a l to h y d r o g e n with similar
yields. T h e a m o u n t o f h y d r o g e n e v o l v e d f r o m c h i t i n o u s
materials c o u l d be e n h a n c e d by e m p l o y i n g acid a n d
alkali p r e t r e a t m e n t (Fig. 3). H o w e v e r , if we a p p l y the
strategy o f h y d r o g e n e v o l u t i o n described h e r e to large
a m o u n t s o f industrial a n d d o m e s t i c wastes, it w o u l d n o t
be feasible to c h e m i c a l l y p r e t r e a t these wastes. It is neces-
sary to b r e e d o r isolate bacterial strains h a v i n g the abil-
ity to efficiently d e g r a d e v a r i o u s b i o p o l y m e r s a n d c o n v e r t
t h e m into h y d r o g e n .
W e a i m to i m p r o v e the h y d r o g e n p r o d u c t i v i t y o f C.
paraputrificum M-21 f r o m wastes by increasing t h e ex-
p r e s s i o n levels o f e n z y m e s i n v o l v e d in the p a t h w a y by
genetic engineering.
ACKNOWLEDGMENTS
We thank Shikishima Starch MFG., Co. Ltd. for supplying the
corn fiber. This work was supported in part by a Grant-in-Aid for
University and Society Collaboration (12794004) from the Ministry
of Education, Science, Sports and Culture, Japan and by a grant
from Aichi Prefecture Joint-Research Project for Regional Inten-
sive, Aichi Science and Technology Foundation, Japan.
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