Journal of Bioscience and Bioengineering

VOL. 124 No. 5, 528e533, 2017

www.elsevier.com/locate/jbiosc

Continuous volatile fatty acid production from lignocellulosic biomass by a novel

rumen-mimetic bioprocess

Hitosi Agematu,1, * Takehiko Takahashi,2 and Yoshio Hamano3

Department of Applied Chemistry, Akita National College of Technology, Akita, Akita 011-8511, Japan,1 Department of Machine Intelligence and Systems Engineering, Faculty of
Systems Science and Technology, Akita Prefectural University, Yurihonjo, Akita 015-0055, Japan,2 and Field Education and Research Center, Faculty of Bioresource Sciences, Akita
Prefectural University, Ohgata, Akita 010-045, Japan3

Received 10 March 2017; accepted 16 June 2017

Available online 6 July 2017
Lignocellulosic biomass is an attractive source of biofuels and biochemicals, being abundant in various plant sources.
However, processing this type of biomass requires hydrolysis of cellulose. The proposed rumen-mimetic bioprocess
consists of dry-pulverization of lignocellulosic biomass and pH-controlled continuous cultivation of ruminal bacteria
using ammonium as a nitrogen source. In this study, ruminal bacteria were continuously cultivated for over 60 days and
used to digest microcrystalline cellulose, rice straw, and Japanese cedar to produce volatile fatty acids (VFAs). The
ruminal bacteria grew well in the chemically defined medium. The amounts of VFAs produced from 20 g of cellulose, rice
straw, and Japanese cedar were 183 ± 29.7, 69.6 ± 12.2, and 21.8 ± 12.9 mmol, respectively. Each digestion completed
within 24 h. The carbon yield was 60.6% when 180 mmol of VFAs was produced from 20 g of cellulose. During the
cultivation, the bacteria were observed to form flocs that enfolded the feed particles. These flocs likely contain all of the
bacterial species necessary to convert lignocellulosic biomass to VFAs and microbial protein symbiotically. Denaturing
gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments revealed that the bacterial com-
munity was relatively stable after 1 week in cultivation, though it was different from the original community structure.
Furthermore, sequence analysis of the DGGE bands indicates that the microbial community includes a cellulolytic
bacterium, a bacterium acting synergistically with cellulolytic bacteria, and a propionate-producing bacterium, as well
as other anaerobic bacteria.
Ó 2017, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Lignocellulosic biomass; Ruminal bacteria; Volatile fatty acids; Rumen-mimetic bioprocess; PCR-denaturing gradient gel
electrophoresis]

The utilization of plant biomass for the production of fuels and
chemicals has become one of the most important avenues of
research in the global shift from fossil fuels to renewable resources.
Lignocellulosic biomass is the most abundantly available plant
biomass, and it is a particularly attractive source because it is non-
edible. To utilize it, the cellulose needs to be hydrolyzed by cellu-
lolytic enzymes or other chemical reactions. However, it is
extremely difficult to degrade cellulose because it is insoluble and
forms very stable hydrogen-bonded crystalline fibers. Since the
cellulolytic enzymes currently available are expensive, other
chemical procedures, including the use of strong acids, high pres-
sure, and high temperature, are generally employed to degrade
cellulose to glucose. Unfortunately, these methods impose a large
environmental burden. Therefore, a process that converts ligno-
cellulosic biomass into useful chemicals economically and with
lower environmental load would be greatly beneficial.
Ruminant animals, such as cattle and sheep, have a diverse and
dense microbial population in their rumen, which forms the largest
compartment of their stomach. The rumen is essential for the
* Corresponding author. Tel.: þ81 18 847 6063; fax: þ81 18 847 6066.
E-mail address: agematu@akita-nct.ac.jp (H. Agematu).
proper digestion of animal feeds containing grain and plant
biomass. All ruminal digestion is performed by the microorganisms
present in the rumen, the diversity of which is known to be highly
responsive to changes in diet, age, and health of the host animal
(1,2). Of these the microorganisms, ruminal bacteria are the most
abundant and are responsible for a considerable portion of the
plant biomass digestion process, leading to the production of vol-
atile fatty acids (VFAs), such as acetic acid, propionic acid, and
butyric acid (3). VFAs are actually considered waste products from
the bacteria; however, ruminal animals absorb them and use them
as a major source of energy (4). Bacteria associated with feed par-
ticles are the most important group for fiber degradation because of
their predominance in terms of bacterial mass and high cellulolytic
activity. Acetic acid has been widely applied in the food industry (as
an acidulant and preservative) as well as the plastic, textile, and
pharmaceutical industries. Propionic acid and butyric acid are also
important chemicals. And furthermore, VFAs are promising sources
of biofuels (5). Using the ruminal bacterial community as fer-
menting organisms makes it possible to convert lignocellulosic
biomass into biobased products in a process termed as a rumen-
mimetic bioprocess. In fact, conversion of lignocellulosic biomass
using ruminal microorganisms has attracted interests among re-
searchers worldwide and various rumen-dominated processes

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2017.06.006
VOL. 124, 2017 CONTINUOUS VOLATILE FATTY ACID PRODUCTION FROM BIOMASS
have been evaluated (6). However, we still need the efforts to
explore and develop novel bioprocesses to utilize abundant organic
resources. In the process, solids/liquids separation is important to
maintain high concentration of substrates and ruminal bacteria in
an anaerobic reactor. Anaerobic sequencing batch reactors (7) and
up-flow anaerobic sludge blanket reactors (8) have been proposed
as reactors with no filtration unit for the separation. In both re-
actors, solids (biomass and ruminal bacteria) are separated from
liquid (product) by sedimentation. Therefore, the sedimentation
rate of solids is important for their industrial application, although
most lignocellulosic biomass floats on liquid at first and agitation of
liquid and solid media is needed for high VFA yields. Another point
for improvement of proposed processes is that it is difficult for
ruminal bacteria to digest a woody biomass. To our knowledge,
there is no report that describes economically production of VFAs
from a woody biomass by ruminal bacteria. We should focus on
developing effective pretreatments to make the cellulose compo-
nent of the biomass more digestible to ruminal bacteria.
In this study, we present a promising and industrially practi-
cable strategy for the bioconversion of lignocellulosic biomass into
useful industrial chemicals. In the process, dry-pulverized ligno-
cellulosic biomass was used for accelerating its digestion and for
easy separation of the biomass and culture solution. The process
also enables VFAs production from a woody biomass. We culti-
vated ruminal bacteria collected from cattle rumen and monitored
the continuous production of VFAs from the digestion of micro-
crystalline cellulose (a chemically defined carbon source), rice
straw (a typical herbaceous soft biomass), and Japanese cedar (a
woody biomass). The diversity of the bacterial community was
also monitored during this process by denaturing gradient gel
electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA
fragments.
MATERIALS AND METHODS
Rumen fluid sampling Beef cows (Japanese Shorthorn) were used in this
study. About 1 L of rumen fluid was anaerobically collected using a stainless steel
oral catheter for cattle (Sanshin Industrial Co. Ltd., Kanagawa, Japan) and was
transferred into a bottle. The bottle was immediately filled with O2-free CO2 gas and
was kept warm at 39 C. The experiment was approved by the Animal Care and Use
Committee at Akita Prefectural University.
Continuous cultivation Approximately 1 L of the rumen fluid was diluted
with the same volume of artificial saliva containing (per liter) 10.6 g of NaHCO3,
0.57 g of KCl, 4.7 g of Na2HPO4$12H2O, 0.12 g of MgSO4$7H2O, 0.04 g of CaCl2$2H2O,
0.56 g of urea, and 1.0 g of (NH4)2SO4 in a fermentation reactor (the working volume
of 2.0 L). The cultivation of ruminal microorganisms was carried out anaerobically by
intermittent ventilation with nitrogen gas produced with a nitrogen gas generator
Model O2B System (Instruments Co., Ltd., Tokyo, Japan) at an incubation tempera-
ture of 39 C. To encourage solideliquid separation in the reactor, the contents were
slowly agitated at 30e40 rpm with two four-bladed impellers. Twenty g of
microcrystalline cellulose (Merck KGaA, Darmstadt, Germany), rice straw powder,
or Japanese cedar powder was added to the reactor every 24 h in each
experiment. The powders were prepared by pulverizing rice straw or woody chips
of Japanese cedar (Cryptomeria japonica D. Don) in a dry condition by a vibration
mill using cog-ring media, called a tandem-ring mill (9). The average particle
diameter of the powder was 20e50 mm.
During cultivation, the pH of the culture solution was recorded and maintained
above 6.50 via the automatic addition of alkaline artificial saliva (described above)
using one channel of a two flow channel peristaltic pump. Simultaneously the same
volume of culture suspension was withdrawn from the reactor as an effluent using
the other channel of the peristaltic pump. The addition of artificial saliva and the
removal of the same volume of culture suspension were regulated by a pH meter-
controller DJ-1023 (ABLE Co., Tokyo, Japan). The fermentation reactor was anaero-
bically operated for over 60 days. To analyze microbial community structure in the
reactor, 10 ml of the reactor contents was collected before daily feeding and was
preserved at 20 C.
Continuous production of VFAs from cellulose and Japanese cedar Tow
fermentation reactors were operated as described above. Ten g of microcrystalline
cellulose and 20 g of Japanese cedar powder were added to each reactor every 24 h
for 32 days. The amount of produced VFAs in the effluent collected every 24 h was
determined and was indicated as integrated value.
529
High-performance liquid chromatography analysis The amounts of acetic
acid, propionic acid, and butyric acid in the effluent were determined using a Nexera
UHPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a diode array ultra-
violet detector set at 210 nm and a InertSustain C18 column (2 mm, 75  2.1 mm i.d.,
GL Science Inc., Tokyo, Japan). A 2 ml high-performance liquid chromatography
(HPLC) sample was chromatographed with an acetonitrile concentration linear-
gradient in 20 mM phosphate buffer (pH 2.5) from 1% (v/v) to 50% (v/v) for 5 min
at a flow rate of 0.3 ml/min at 40 C. The HPLC sample was prepared as follows:
200 ml of 200 mM phosphate buffer (pH 2.5) was added to 200 ml of the
supernatant of the effluent. The mixture was centrifuged at 15,000 g for 5 min
and the supernatant was used for HPLC analysis.
Bacterial DNA extraction A total of 10 ml of frozen reactor content was
defrosted and added to 0.2 g of glass beads (diameter 500e710 mm). The samples
were shaken for 1 min using a vortex mixer and then incubated on ice to separate
the liquid phases from the solid phases. Total DNA was extracted from the liquid
phases using an ISOPLANT II DNA extraction kit (Nippon Gene Co., Ltd., Tokyo, Japan)
according to the DNA extraction protocol provided by the manufacture. The quantity
and quality of the extracted DNA were verified by measuring the absorbance at 260
and 280 nm using a spectrophotometer, and the final concentration of DNA was
adjusted to 10 ng/ml for PCR-DGGE analysis.
16S rDNA PCR-amplification From the DNA extract, bacterial 16S rDNA was
amplified using the primer pairs GC-357F (5ʹ-CGCCCGCCGCGCGCGG
CGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3ʹ) and 518R (5ʹ-
GTATTACCGCGGCTGCTGG-3ʹ) (10). A reaction mixture containing 20 pmol of each
primer, 50 ng of template DNA, and 20 ml of Emerald Amp PCR master mix
(Takara Bio Inc., Shiga, Japan) in a total volume of 40 ml was prepared. PCR was
performed by GeneAtlas 322 (ASTEC Co. Ltd., Fukuoka, Japan) using the following
temperature program: an initial denaturation at 94 C for 5 min, 94 C for 1 min,
decreasing temperature gradient from 65 to 55 C by 0.5 C each cycle for 1 min,
72 C for 1 min for 20 cycles, followed by 10 cycles of 1 min denaturation at 94 C,
1 min annealing at 55 C, and 1 min extension at 72 C with a final extension at
72 C for 7 min. The amplicons were examined by electrophoresis on 2.0% (w/v)
agarose gel.
DGGE analysis A total of 5 ml of each PCR-amplified 16S rDNA sample was
loaded on to an 8% polyacrylamide gel with TAE buffer (20 mM Tris-acetate,
10 mM sodium acetate, 0.5 mM EDTA, pH 8.0) and a 30e60% linear gradient of
denaturant of urea and formamide (100% denaturant corresponded to 40% (v/v)
deionized formamide and 7 M urea). Electrophoresis was performed in a Bio-Rad
DCode system (Bio-Rad Laboratories, Inc., CA, USA) at 60 C and 130 V for 5 h.
DGGE Marker III (Nippon Gene Co. Ltd.) was loaded in to lanes at both ends of
each gel. Gels were stained with SYBR Green I (Takara Bio) according to
manufacturer’s instructions and photographed with UV transillumination using a
Gel Doc EZ System (Bio-Rad Laboratories).
Sequence analysis Prominent DGGE bands were cut from the gel and the gel
pieces were crushed in 50 ml of TE buffer (10 mM TriseHCl, 1 mM EDTA, pH 8.0). DNA
fragments from the DGGE bands were purified using a NucleoSpin Gel and PCR
Clean-up Kit (Macherey-Nagel Inc., PA, USA). The purified DNA was used as the
template for PCR amplification as described above only using a 357F primer
without a GC clamp. The PCR products were purified using a NucleoSpin Gel and
PCR Clean-up Kit and were send to Biotechnology Center of Akita Prefectural
University for nucleotide sequencing analysis. The analyzed partial 16S rDNA
sequences were compared with sequences in the GenBank database using the
NCBI BLAST search program.
RESULTS
Design of the rumen-mimetic bioprocess Pulverized rice
straw and Japanese cedar could be separated from culture fluid by
sedimentation because they sunk in water. The Japanese cedar
powder was digested by ruminal bacteria, while the sawdust of it
was not digested at all. The results show that pulverization of a
substrate accelerates its digestion by ruminal bacteria. The ruminal
bacteria were continuously cultivated for over 60 days under
anaerobic conditions and used to digest lignocellulosic biomass to
produce VFAs. VFAs production was monitored with a pH meter,
which allowed the digestion activity of the ruminal bacteria to be
estimated as the pH value decreased. The composition of the arti-
ficial saliva is fundamentally similar to that of sheep and calf (11)
except for the addition of ammonium sulfate. The optimum
concentration of ammonium sulfate in the artificial saliva was
0.1% (w/v). The supply of ammonium was replenished via the
addition of artificial saliva as the pH decreased, a process that
was controlled by a pH meter-controller. After a few days of the
530 AGEMATU ET AL. J. BIOSCI. BIOENG.,

FIG. 2. Typical pH pattern observed during ruminal digestion of cellulose. Microcrys-

talline cellulose (20 g) was added, and the pH was recorded in 20-minute intervals. The
pH was controlled so as not to decrease below 6.50 via the automatic addition of the
artificial saliva.

69.6  12.2, and 21.8  12.9 mmol, respectively (Table 2). Each
digestion was completed within 24 h.

Continuous production of VFAs from cellulose and Japanese

FIG. 1. Representative images of the flocs of ruminal bacteria formed during continuous
cultivation. Microscopic observation of the flocs that enfolded some feed particles.
Scale Bar: 200 mm (A), 50 mm (B).
cultivation, the ruminal bacteria formed flocs that appeared to
enfold the feed particles (Fig. 1). The reactor contents were slowly
agitated at 30e40 rpm to help prevent the flocs from flowing out
of the reactor. Protozoa disappeared from the culture solution at
an early stage of the cultivation.
cedar Microcrystalline cellulose was chosen as a substrate to
show that the process is applicable to a substrate not containing
any nutrients except for a carbon source and that the ruminal
bacteria can be continuously cultivated in a chemically defined
medium in this process. Japanese cedar powder was also chosen to
show that the pulverization is essential to steady production of
VFAs from a woody biomass. To approximately equalize the cellu-
lose content of both substrates, 10 g of cellulose and 20 g of Japa-
nese cedar powder were added to the reactors every 24 h because
TABLE 1. Production of acetic acid, propionic acid, and butyric acid from 20 g of
cellulose by the rumen-mimetic bioprocess.

VFA mMa mmolb Carbon (mmol)c Carbon (%)d g

VFAs production from cellulose and lignocellulosic
Acetic acid 60.2 100.6 201.2 27.2 6.04
biomass When 20 g of microcrystalline cellulose was added to
Propionic acid 42.5 71.0 213.0 28.8 5.26
the reactor, the pH of culture solution immediately decreased Butyric acid 5.16 8.6 34.4 4.6 0.76
because of VFAs production (Fig. 2). It is known that ruminal Total VFAse 107.9 180.2 448.6 60.6 12.1
bacteria rapidly attach to recently ingested feed particles (12). Cellulose 740.0 100 20
After 1 h, the pH was adjusted to 6.50 by addition of artificial a
The concentration of VFAs in the effluent (1670 ml).
b
saliva. It was often difficult to maintain the fermentation pH at The amount of VFAs.
c
6.50 and the pH temporarily decreased below 6.50 when the The amount of carbon in the VFAs.
d
The carbon yield (%) when 20 g cellulose was added.
VFAs production rate was high. After 24 h, the pH exceeded 6.50 e
Total VFAs is the combined amount of acetic acid, propionic acid, and butyric
because VFAs production ended. In this experiment, 1670 ml of acid.
effluent was extracted for 24 h. The amounts of acetic acid,
propionic acid, and butyric acid in the effluent were determined
by HPLC analysis and are summarized in Table 1. Acetic acid, TABLE 2. Production of acetic acid, propionic acid, and butyric acid from 20 g of each
propionic acid, and butyric acid were the primary VFAs in the substrate using the rumen-mimetic bioprocess.

effluent, whereas lactic acid was not produced. A total of Substrate Weight (g) Production (mmol)
180 mmol of VFAs was produced from 20 g of cellulose. The Acetic acid Propionic acid Butyric acid Total VFAsa
carbon yield was 60.6% and was calculated with the following
Cellulose 20 90.3  10.9 83.5  21.9 9.0  4.5 183  29.7
equation: carbon yield (%) ¼ value (mole) of carbon in produced Rice straw 20 37.7  7.7 26.9  5.3 4.9  0.9 69.6  12.2
total VFAs/0.74 (mole, value of carbon in 20 g of cellulose)  100. Japanese cedar 20 8.9  5.3 11.9  7.1 1.0  0.6 21.8  12.9
VFAs were also produced from rice straw and Japanese cedar Values represent means  SE (n ¼ 4).
using the same procedure. The amounts of VFAs produced from a
Total VFAs is the combined amount of acetic acid, propionic acid, and butyric
20 g of cellulose, rice straw, and Japanese cedar were 183  29.7, acid.
VOL. 124, 2017 CONTINUOUS VOLATILE FATTY ACID PRODUCTION FROM BIOMASS
FIG. 3. Continuous production of VFAs from cellulose and Japanese cedar. The amounts
of produced VFAs were indicated as integrated values. Symbols: open circles, micro-
crystalline cellulose; closed circles, Japanese cedar powder.
the typical cellulose content of wood is 45.1% (w/w) (13). VFAs were
continuously produced from these substrates for 32 days (Fig. 3).
The amounts of the produced VFAs are summarized in Table 3.
The VFAs production rate from microcrystalline cellulose and
Japanese cedar powder were 56.7 and 23.0 mmol/d, respectively.
The average volumes of the effluents from the reactors were 670
and 295 ml/d, respectively, meaning that the dilution rates were
0.335 and 0.148 d1, respectively.
PCR-DGGE analysis of microbial community
structure Another objective of this study was to detect
changes in the microbial community during continuous cultivation
compared to that of the original bovine rumen fluid. To do so, the
microbial community structure of the 60 day cultivation was
analyzed by PCR-DGGE (Fig. 4). The band profile shown in the DGGE
gels reflects the predominant bacterial species in the samples.
Visual comparison of the banding patterns indicates that a
relatively stable community that is distinct from the initial
community structure was formed after 1 week in cultivation,
although the strength of a band changed. Fig. 4 shows that some
prominent bands (bands 1 to 9) appeared in multiple lanes.
Therefore, these 9 major DGGE bands were collected and
sequenced. These sequences have been submitted to the DDBJ/
EMBL/GenBank databases under accession numbers
LC199890eLC199898. Table 4 shows the closest relatives of each
band according to the BLAST sequence analysis (14e22). The 16S
rDNA sequence of band 7 shows high similarity with that of
TABLE 3. Production of acetic acid, propionic acid, and butyric acid from 320 g of
microcrystalline cellulose and 640 g of Japanese cedar powder using the rumen-
mimetic bioprocess.
Substrate Weight (g) Production (mol)
Acetic acid Propionic acid Butyric acid Total VFAsa
Cellulose 320 1.17 0.596 0.047 1.81
Japanese cedar 640 0.447 0.128 0.121 0.695
a
Total VFAs is the combined amount of acetic acid, propionic acid, and butyric
acid edar using the rumen-mimetic bioprocess.
531
Succiniclasticum ruminis SE10 (98%) isolated from rumen.
Prevotella ruminicola Bryant 23 (93%) and Fibrobacter succinogenes
HM2 (89%), the relatives of bands 2 and 6, respectively, were also
previously isolated from rumen. Almost all of the strains listed in
Table 4 can grow anaerobically.
DISCUSSION
In this study, we focused on the utilization of the bovine ruminal
bacteria for the economical production of VFAs from lignocellulosic
biomass, naming this process a rumen-mimetic bioprocess. The
process provides a simple operation of the ruminal fermentation:
(i) The pulverization of lignocellulosic biomass increases the rate of
cellulose fermentation and facilitates successful separation of solids
(biomass and flocs) and liquid (fermentation products); (ii) The pH-
controlled continuous cultivation is an automatic operation to
provide all of the nutritional requirements of the bacterial popu-
lation except for a carbon source and to remove fermentation
products; (iii) Utilization of ammonium in the artificial saliva
means that this process is applicable to any biomass not containing
sufficient crude protein like wood and paper. In ruminants, initial
chewing (mastication) of the feed immediately after intake and
rumination of partially digested feed are important to accelerate
the digestion of feed. In a biorefinery, however, mechanical pul-
verization of biomass has generally been considered impractical
and too expensive (23). The tandem-ring mill we used in this study
is an energy saving mill developed for effective pretreatment of
lignocellulosic biomass (9). Pulverized biomass and the flocs can be
separated from liquid by sedimentation (i.e., a clarifier tank),
meaning that the process can maintain high concentration of
biomass and the cellulolytic bacteria in the reactor.
The carbon yields from cellulose in Tables 2 and 3 were 63.1%
and 36.4%, respectively, and were not the same value. The reason is
considered to be that the supply of ammonium as a nitrogen source
was a limiting factor of the growth (anabolism) of ruminal bacteria
in the continuous production of VFAs from cellulose (Table 3) and
so 10 g of cellulose supplied a day was not always consumed
completely.
The typical cellulose content of rice straw and wood are 39.2%
(w/w) and 45.1% (w/w) (based on dry weight), respectively (13). In
this study, the amount of VFAs produced from 20 g of Japanese
cedar was lower than that produced from 20 g of rice straw
(Table 2), even though wood contained more cellulose than rice
straw. One possible explanation for this result is that the cellulose
of the Japanese cedar is more tightly covered with lignin than that
of rice straw, making it more difficult for the cellulolytic bacteria to
access. The VFAs production rate of microcrystalline cellulose and
Japanese cedar powder were 5.67 and 2.55 mmol/g-cellulose/d,
respectively (Fig. 3). This result shows that the accessibility of the
cellulolytic bacteria to the cellulose of Japanese cedar increased
from 0% (sawdust) to 45% by the pulverization if the accessibility to
microcrystalline cellulose was presumed to be 100%. During the
conversion of cellulose to VFAs, the rate-limiting reaction is the
hydrolysis of cellulose and altering its accessibility could advance
the process.
During our experiments, the ruminal bacteria were observed to
form anaerobic aggregates, or flocs, which are a special type of
biofilms. The flocs appeared to engulf the feed particles. Forming
flocs is advantageous to make full use of the synergic effect of
ruminal bacteria. Notably, adhesion of ruminal bacteria to the plant
fibers is an important step in the subsequent digestion of the
lignocellulosic biomass. Bacteria associated with feed particles are
the major component (70%) of the total bacterial population (24,4).
It is likely that the flocs formed in this bioprocess because the feed
particles were enough small for the ruminal bacteria to enclose
532 AGEMATU ET AL. J. BIOSCI. BIOENG.,

FIG. 4. DGGE profile of bacterial 16S rDNA in the microbial community of the continuous cultivation. The time course (in days) of the cultivation is indicated above each lane. Bands 1
to 9 were selected for sequencing. M: DGGE Maker Z.

TABLE 4. The closest relatives of the bacterial 16S rDNA sequences from the DGGE bands 1e9.

Band No. Accession no. The closest relative (accession no.) Identity Source (growtha) Reference

1 LC199890 Chryseobacterium marinum NBRC 103143 (NR_114212.1) 83 Seawater (a) 14

2 LC199891 Prevotella ruminicola Bryant 23 (NR_102887.1) 93 Rumen (b) 15
3 LC199892 Dysgonomonas hofstadii JCM 17038 (NR_113230.1) 84 Human (b) 16
4 LC199893 Capnocytophaga sputigena JCM 12967 (NR_113564.1) 86 Periodontal lesions (b) 17
5 LC199894 Mycoplasma yeatsii GIH (NR_026037.1) 84 Ear canals of goat (a, b) 18
6 LC199895 Fibrobacter succinogenes HM2 (NR_104844.1) 89 Rumen of sheep (b) 19
7 LC199896 Succiniclasticum ruminis SE10 (NR_026205.1) 98 Rumen of cow (b) 20
8 LC199897 Sedimentibacter hydroxybenzocus JW/Z-1 (NR_029146.1) 91 Freshwater sediment (b) 21
9 LC199898 Mucilaginibacter composti TR603 (NR_112621.1) 84 Compost (a) 22
a
a, aerobic growth; b, anaerobic growth.

them. It is also likely that the flocs included all of the ruminal
bacterial species necessary to convert cellulose and ammonium
into VFAs and microbial protein (cells), and that these bacteria have
a symbiotic relationship with each other. For example, S. ruminis,
the relative (98%) of band 7, has been shown to metabolize succi-
nate to propionate, while not altering any other energy sources,
such as carbohydrates, pyruvate, lactate, dicarboxylic acids, aspar-
tate, citrate, and trans-aconitate (20), indicating that this organism
likely has a symbiotic relationship with other ruminal organisms.
F. succinogenes and P. ruminicola produce succinate as a fermenta-
tion product from pyruvate via malate (4,25,26). However, succi-
nate did not accumulate in the reactor. S. ruminis is considered to
have a symbiotic relationship with F. succinogenes and P. ruminicola.
F. succinogenes, the relative (89%) of band 6, is a cellulolytic
bacterium that degrades plant cell wall biomass in ruminant ani-
mals and is among the most rapidly fibrolytic of all mesophilic
bacteria (27). Digestion of cellulose by this organism requires the
attachment of the cells to the cellulose fibers (28,29). Therefore,
access and attachment to the cellulose is essential during the
digestion process for the cellulolytic bacteria. Interestingly, the
genus Fibrobacter is known to have broad genetic diversity, as
highlighted by a previous DNA hybridization and 16S rDNA simi-
larity analysis (30). Therefore, the bacterium corresponding to band
6 is presumed to be of the genus Fibrobacter.
P. ruminicola, the relative (93%) of band 2, contributes to plant
cell wall degradation by acting synergistically with cellulolytic
bacteria (31). This organism is relatively abundant in rumen bac-
teria, and previous real-time PCR quantification of this genus
indicated that it makes up approximately 19.7% of the total rumen
bacteria population (32). However, the known Prevotella species
only account for 2e4% of the total bacterial 16S rRNA gene copies
(33). These results suggest that the majority of Prevotella in the
rumen cannot be brought into pure culture. The present study
demonstrates that the bacterium corresponding to band 2 is likely
of the genus Prevotella.
In conclusions, the pulverization of lignocellulosic biomass is
essential to the rumen-mimetic bioprocess. The increase in surface
area of substrates is the key factor regulating the rate of cellulose
fermentation because the accessibility of cellulolytic bacteria to the
cellulose of substrates is the rate-limiting step of their digestion.
This study elucidated the effect of the pulverization by using pul-
verized Japanese cedar as a substrate. It is necessary to further
develop the pulverization process corresponding to biomass pre-
treatment for the economical production of VFAs. Another impor-
tant point is the microbial community structure of ruminal bacteria
in the process. It may be possible to increase the digestion rate of
lignocellulosic biomass by the introduction of a bacterium having a
higher cellulolytic activity to the ruminal microbial community. For
this purpose, the overall analysis of the microbial community
structure of the flocs has been continuing.
ACKNOWLEDGMENTS
We thank Dr. Yoichiro Shimura (Akita Prefectural University) for
technical advice on PCR-DGGE. Tshis work was supported by a grant
from JSPS KAKENHI (grant number: 15K14701).
VOL. 124, 2017 CONTINUOUS VOLATILE FATTY ACID PRODUCTION FROM BIOMASS
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