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Biochemical and Biophysical Research Communications 327 (2005) 884893


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Evaluation of support matrices for immobilization of anaerobic


consortia for ecient carbon cycling in waste regeneration
Ashvini Chauhan*, Andrew Ogram
Soil and Water Science Department, Soil Molecular Ecology Laboratory, University of Florida, P.O. Box 110290, Gainesville, FL 32611-0290, USA
Received 10 December 2004
Available online 24 December 2004

Abstract
Ecient metabolism of fatty acids during anaerobic waste digestion requires development of consortia that include fatty acid
consuming H2 producing bacteria and methanogenic bacteria. The objective of this research was to optimize methanogenesis from
fatty acids by evaluating a variety of support matrices for use in maintaining ecient syntrophicmethanogenic consortia. Tested
matrices included clays (montmorillonite and bentonite), glass beads (106 and 425600 lm), microcarriers (cytopore, cytodex, cytoline, and cultispher; conventionally employed for cultivation of mammalian cell lines), BioSep beads (powdered activated carbon),
and membranes (hydrophilic; nylon, polysulfone, and hydrophobic; teon, polypropylene). Data obtained from headspace methane
(CH4) analyses as an indicator of anaerobic carbon cycling eciency indicated that material surface properties were important in
maintenance and functioning of the anaerobic consortia. Cytoline yielded signicantly higher CH4 than other matrices as early as in
the rst week of incubation. 16S rRNA gene sequence analysis from crushed cytoline matrix showed the presence of Syntrophomonas spp. (butyrate oxidizing syntrophs) and Syntrophobacter spp. (propionate oxidizing syntrophs), with Methanosaeta spp. (acetate utilizing methanogen), and Methanospirillum spp. (hydrogen utilizing methanogen) cells. It is likely that the more hydrophobic
surfaces provided a suitable surface for adherence of cells of syntrophicmethanogenic consortia. Cytoline also appeared to protect
entrapped consortia from air, resulting in rapid methanogenesis after aerial exposure. Our study suggests that support matrices can
be used in anaerobic digestors, pre-seeded with immobilized or entrapped consortia on support matrices, and may be of value as
inoculant-adsorbents to rapidly initiate or recover proper system functioning following perturbation.
 2004 Elsevier Inc. All rights reserved.
Keywords: Support matrices; Immobilization; Wastewater; Syntrophic bacteria; Methanogens

Anaerobic wastewater treatment systems are used for


treating a wide range of industrial euents, including
those containing toxic or inhibitory compounds [1,2].
The process is also used for treatment of domestic
wastewater and is commonly applied for purication
of industrial wastewaters with high organic matter content. Aerobic digestors are in wider use than anaerobic
systems, even though they require higher amounts of
capital resources and skilled manpower. In aerobic processes, approximately 67% of the organic matter is converted to cell biomass, which has signicant running
*

Corresponding author. Fax: +1 352 392 3902.


E-mail address: ashvini@u.edu (A. Chauhan).

0006-291X/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.12.083

costs due to daily sludge production requiring further


treatment and safe disposal. In contrast to this, anaerobic digestion converts approximately 3% of the wastewater organic matter to cell biomass with much of the
remaining carbon being converted to CH4 and CO2
gases as stable-end products. In anaerobic systems, biological sludge production is much lower than in aerobic
systems, further reducing the costs of treatment and disposal [3,4]. Finally, CH4 can be utilized as a value-added
product for generation of energy [2].
A complex interdependent community comprised of
hydrolytic, fermentative, syntrophic, homoacetogenic,
and methanogenic microorganisms recycles carbon in
anaerobic waste reclamation systems [1,5]. Generation

A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

of CH4 depends on at least three interacting groups of


microbes. Cellulolytic and fermentative bacteria decompose cellulose and other complex biomolecules to alcohols and fatty acids (e.g., propionate, butyrate), CO2,
and H2. Interspecies H2 or formate transfer between
syntrophic bacteria and methanogens further converts
alcohols and fatty acids into acetate, CO2, and H2,
which serve as carbon and electron donors for methanogens to form CH4 [1,5,6]. Fatty acids formed in these
series of reactions by primary fermentors are potent
thermodynamic inhibitors and require coordinated consumption by syntrophic bacteria for optimum growth
[1,5,6].
Immobilization of microbial consortia is important
for optimum functioning of anaerobic treatment systems, such that concentrations of intermediates are suciently low for ecient carbon cycling. It has been
reported that a distance of less than 1 lm between syntrophic bacteria and methanogens is a prerequisite for
oxidation of fatty acids, allowing transfer of reducing
equivalents to methanogens [5]. Hydrogen and acetate
levels should be below threshold limits so that these reactions are exergonic and proceed to completion [7,8]. Poor
coupling of syntrophs with methanogens results in
accumulation of H2 and acetate, and reaction(s) become
endergonic. H2 and acetate may accumulate, which decreases pH and leads to collapse of the system [1,9,10].
The addition of support materials, such as sepiolite,
to uidized-bed anaerobic digesters has been shown to
enhance CH4 production by potentially increased colonization by syntrophic microbiota [1113]. The objective
of this study was to evaluate a variety of support matrices to immobilize fatty acid oxidizing-hydrogen producing syntrophic bacteria and methanogens, which may
expedite carbon cycling. A range of entrapment matrices
and biolm supports are available for use in such applications [1115]. However, to our knowledge, there are
no reports that have assessed eects of support matrices
on rates of fatty acid metabolism to CH4.
An additional objective was to investigate the eect of
immobilization of syntrophicmethanogenic consortia
and their use in perturbed conditions. Problems may
arise during storage and transport, including loss of
activity by key bacterial groups in the starting sludge
[9,16]. The existing microbial community may not be
adapted to conditions in a new reactor or the presence
of toxic compounds [17]. Therefore, use of active consortia immobilized in suitable matrices may be useful
for rapid startup or recovery of reactor functioning.
Materials and methods
Consortia isolation, microcosm construction, and growth conditions.
An anaerobic consortium (termed as F1 consortium) was isolated from
eutrophic soils from the Florida Everglades [18]. Serum bottles containing 25 ml of anaerobically prepared basal carbonate yeast extract

885

trypticase medium (BCYT) [19] were crimped using rubber septa with
aluminum seals (Bellco Glass, Vineland, N.J.). Fatty acids (20 mM
propionate or butyrate) were added from anaerobically prepared stock
solutions, resazurin (0.1%) was added as redox indicator and reduced
with cysteinesulde solution as previously described [18]. F1 consortia
were added to a nal volume of 5% by nitrogen-ushed syringes to
avoid aerial contact and incubated at 30 C in the dark for syntrophic
methanogenic associations to develop. Subsequent enrichments were
transferred to BCT medium (basal carbonate trypticase without yeastextract) to discourage background growth of primary fermentors.
Standard anaerobic culturing techniques were followed [20].
To test the tolerance of the consortium bound to dierent matrices
on exposure to air, all microcosms containing each of 13 matrices were
spiked with propionate and butyrate. Methanogenesis and depletion of
fatty acids were followed until CH4 production and fatty acid depletion (data not shown) stabilized. This was typically within 2 weeks.
Support matrices were harvested by opening the anaerobic vials,
exposing them to air, and transferring into fresh medium containing
fatty acids.
Cell immobilization/entrapment matrices. Support matrices were
prepared according to the individual matrix and their respective surface areas, and added into 25 ml BCYT medium. Propionate and
butyrate well-grown F1 consortium was centrifuged in an anaerobic
glove box and concentrated 5 times. Five percent of this concentrated
cell-suspension was then added into each anaerobic vial (OD600 was
kept constant at 0.3).
Four types of microcarriers were tested as supports and were
prepared according to the manufacturers instructions (Pharmacia
Biotechnology, St. Albans, UK), washed twice in phosphate-buered
saline (150 mM sodium phosphate; 150 mM NaCl, pH 7.2), and added
in the medium. Microcarriers (initially used at 3.8 g cytoline 2; 75 mg
cytopore 2; 720 mg cytodex 3, and 25 mg cultispher each per 25 ml), as
previously described [21], were sterilized by autoclaving. BioSep beads
consist of powdered activated carbon (PAC) and are 34 mm in
diameter. These consist of 25-wt% polymers and 75-wt% PAC [22,23],
and were pretreated by incubating at 300 C for 3 h and 1 g was added.
Six ltration membranes circles, 25 mm in diameter, were introduced
into each serum bottle prior to inoculation (hydrophobic membranes,
polypropylene and teon; hydrophilic membranes, nylon and polysulfone). Additionally, fatty acids were spiked onto the surface of
hydrophilic membranes, allowed to dry, and introduced into anaerobic
bottles under anaerobic conditions. Two dierent sizes of glass beads
(106 and 425600 lm) 600 and 50 mg of clays bentonite (average pore
size > 2 lm; Sigma, St. Louis, MO) and magnesium montmorillonite
(SM1200, 200 mesh; GSA Resources, Cortario, Arizona) were also
tested. All support matrices were saturated with BCYT medium as
outlined above and tested for methanogenesis.
Biomass measurements. Bacterial biomass on the support materials
was quantied by a modication of the Biuret method for protein
determination [21]. Every week one serum bottle from each support
matrix was sacriced and the matrix was placed in a test tube with 1 ml
of 3 M NaOH. Samples were boiled for 10 min and left to cool for
25 min followed by the addition of CuSO4 5 H2O solution (2.5% w/v)
and incubated at room temperature for 5 min. Centrifuged microbial
protein concentrations were determined by a total protein estimation
kit according to the enclosed instructions (Sigma, St. Louis, MO).
Absorbance of the resulting samples was read at 725 nm, and concentrations of protein were determined by comparison with bovine
serum albumin standards against blanks prepared with distilled water.
Analyses were conducted in triplicate.
Analytical methods. Dierential interference contrast (DIC)
microscopy was conducted with a short distance condenser and 100
objective in a Nikon OPTIPHOT (Nippon-Tokyo, Japan) biological
microscope as described [18].
Fatty acids were quantied by HPLC equipped with a UV detector
at 210 nm (Waters, Milford, MA) as outlined earlier [18]. Headspace
pressures were measured prior to gas analysis using a digital pressure

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A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

indicator (DPI 705; Druck, New Faireld, CT). Potential rates of CH4
production by entrapped/immobilized consortia were studied by gas
chromatography with a ame ionization detector in a Shimadzu 8A
GC as described previously [24]. All determinations were carried out in
triplicate.
Nucleic acid extraction and PCR amplication. DNA from enriched
matrices was extracted using UltraClean Soil DNA kits (MoBio,
Solana Beach, CA) with slight modications [18]. DNA was extracted
from pellets of 10 ml microcosms and 0.5 mg of crushed support matrix after 4 weeks of incubation or more depending on methanogenesis
rates.
Primers used for PCR amplication of 16S rRNA gene sequences
were 27F (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ) and 1492R (5 0 TACGGYTACCTTGTTACGACTT-3 0 ) [25]. Archaeal 16S rRNA
gene sequences were amplied with the universal primer 1492R and
Archaea-specic primer 23F (5 0 -TGCAGAYCTGGTYGATYCTG
CC-3 0 ) [26]. All amplications were performed in a GeneAmp PCR
system 2400 (PerkinElmer Applied Biosystems, Norwalk, CT) using
HotStarTaq Master Mix (Qiagen, Valencia, CA), as reported earlier
[27]. PCR products (5 ll) were analyzed by electrophoresis through a
1% agarose gel in TAE buer.
Cloning of bacterial and archaeal 16S genes and RFLP analyses.
Cloning of bacterial and archaeal genes was carried out with fresh
PCR amplicons obtained with appropriate primer sets. Amplicons
were ligated into pCRII-TOPO cloning vector and transformed into
Escherichia coli TOP10F 0 cells according to the manufacturers
instructions (Invitrogen, Carlsbad, CA). Individual colonies of E. coli
were screened by direct PCR amplication with appropriate primers
using previously described PCR programs [27]. Restriction fragment
length polymorphism (RFLP) analyses were conducted using restriction enzymes HhaI and AluI, and were analyzed in 2% agarose gel.
Clone libraries were subjected to rarefaction using aRarefactWin
(version 1.3; S. Holland, Stratigraphy Lab, University of Georgia,
Athens http://www.uga.edu/~strata/software/) to conrm that sucient numbers of RFLP groups were selected to represent the clone
libraries from dierent support matrices.
DNA sequencing and phylogenetic analysis. Selected unique and
common clones after comparison by RFLP were sequenced at DNA
Sequencing Core Laboratory at the University of Florida with 27F and
23F primers. Chimera detection was carried out by Chimera_Check
version 2.7 of the RDP-II website [28]. Sequences were also compared
with previously identied sequences in the National Center of Biotechnology Information (NCBI) database using BLAST [29] and were
aligned by ClustalX v. 1.8 [30]. Phylogenetic trees were generated with
PAUP v. 4.0b8 and rRNA trees were constructed using maximum
parsimony with default settings (D.L. Swoord, Sinauer Associates,
Sunderland, MA). Bootstrap resampling analysis for 100 replicates
was performed to estimate the condence of tree topologies.

Nucleotide sequence accession numbers. The partial 16S rRNA gene


sequences obtained in this study have been deposited in GenBank
under Accession Nos. from AY496074 to AY496078, and AY496250.

Results and discussion


Methanogenesis by consortia
Table 1 lists the physico-chemical properties of the 13
support matrices evaluated for colonization by syntrophicmethanogenic consortia. These matrices and
the concentrations used were chosen based on previous
reports for their use to immobilize/entrap bacterial cells
[11,13,2123]. In this study, dierent matrices represented a range of surface characteristics, including degree of hydrophobicity, and porosity. Most bacterial
cell surfaces are hydrophobic [31,32], and it was hypothesized that hydrophobic matrices would be better adherents for the consortia, and potentially reduce
interbacterial distances, thereby producing greater
amounts of CH4 from propionate and butyrate than less
hydrophobic or porous matrices. These results are
shown in Fig. 1.
Figs. 1A and B show total weekly methanogenesis
from clays (montmorillonite and bentonite), acidwashed glass beads (diameters of 106 and 425
600 lm), and BioSep beads. The general trend observed
for both propionate- and butyrate-induced methanogenesis was montmorillonite > glass beads > bentonite > BioSep beads. Clays such as montmorillonite,
sepiolite, saponite, bentonite, and zeolite have been previously used to immobilize microorganisms to increase
methanogenesis [11]. Increased methanogenesis by
immobilized methanogens was attributed to the porosity
and physico-chemical characteristics of these support
materials, which promote adherence of microorganisms
[33]. Sanchez et al. [12] observed a dependence of methanogenesis on particle size of support material, such that
more CH4 production was observed with smaller particle size. A similar trend was observed in our study, in

Table 1
Characteristics of dierent support matrices employed for entrapment/immobilization of syntrophicmethanogenic assemblages
Matrix

Type

Relative surface
characteristics

Glass beads

Particle sizes 106


and 425600 lm
Pore size 0.22 lm
Pore size 0.22 lm
Pore size 0.22 lm
Pore size 0.22 lm
Pore size 30 lm
Particle size 175 lm
Pore size 10400 lm
Particle size 20 lm
Particle size 200 mesh
Pore size 2 lm
Pore size 1.9 lm

Hydrophilic

Polypropylene (membrane)
Teon (membrane)
Nylon (membrane)
Polysulfone (membrane)
Cytopore-2 (microcarrier), cellulose with positively charged N,N,-diethylaminoethyl groups
Cytodex-3 (microcarrier), dextran beads coated with gelatin
Cytoline-2 (microcarrier), polyethylene (hydrophobic) and silica (negative charge)
CultiSpher-S (microcarrier), cross-linked gelatin matrix
Montmorillonite clay
Bentonite clay
BioSep beads, composed of 25% polymer and 75% powdered activated carbon

Hydrophobic
Hydrophobic
Hydrophilic
Hydrophilic
Hydrophilic
Hydrophilic
Hydrophobic
Hydrophilic
Hydrophilic
Hydrophilic
Hydrophilic

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Fig. 1. Immobilization/entrapment of syntrophicmethanogenic consortium on dierent support matrices spiked with 20 mM propionate (P) or
butyrate (B). CH4 in the headspace was quantied weekly. Matrices were pretreated as outlined in Materials and methods. (A,B) Methanogenesis by
consortia adhered to bentonite, montmorillonite, glass beads (106 and 425600 lm), and BioSep beads. (C,D) Microcarriers (cytopore, cytoline,
cultispher, and cytodex) with dierent surface properties and porosities. (E,F) Hydrophobic (teon and polypropylene) and hydrophilic (nylon and
polysulfone) membranes.

which elevated methanogenesis was observed with


montmorillonite and the smaller glass beads (Figs. 1A
and B).
Microcarriers were also assessed for methanogenesis
potential (Figs. 1C and D). These matrices are generally
used to provide anchorage for culturing mammalian cell
lines, and have dierent coatings on their surfaces
including cross-linked cellulose, dextran, silica, and gelatin. Cytoline yielded maximum methanogenesis from
both fatty acids tested, followed by cytopore and cultispher, which were equally ecient. Cytodex yielded
the least amount of CH4 from both fatty acids. It is
likely that the consortia adhere to cytoline much better
than other matrices because it is coated with polyethylene, which is more hydrophobic relative to other matrices (Table 1). Syntrophic bacteria and methanogens
possess hydrophobic cell surfaces and are expected to
adhere well to hydrophobic matrices [31,32].
Greater hydrophobicity of surface promotes cell-tocell interaction between bacterial consortia, thereby

enhancing the formation of aggregates [34]. Previous research has demonstrated that hydrophobic bacteria tend
to attach better to hydrophobic than to hydrophilic surfaces [35,36]. In further investigations, we compared
hydrophilic (nylon and polysulfone) and hydrophobic
(polypropylene and teon) membranes as adherents
(Table 1). Additionally, hydrophilic membranes onto
which fatty acids were spiked were tested (Figs. 1E
and F). Hydrophobic membranes supported more rapid
methanogenesis, and it is likely that syntrophicmethanogenic consortia adhere better to hydrophobic membranes (polypropylene and teon) than to hydrophilic
membranes (nylon and polysulfone). Based on these
data, it can be concluded that the use of selected support
matrices such as cytoline may be a crucial component in
the optimal functioning of a bioreactor. Bioreactors may
be either pre-seeded by a support matrix harboring
anaerobic consortia, or support matrices may be used
to jumpstart a malfunctioning or failed bioreactor, functioning to decrease the bacterial acclimation process.

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Bioreactor startup is frequently hindered by low microbial growth rates and limited capability of methanogens
to adhere and form xed biolms [16].

of known syntrophic bacteria in the database, indicating


that they represent novel lineages.
Archaeal phylogenetic analysis

Microscopic analysis of high methanogenesis yielding


entrapped consortia
Dierential interference contrast (DIC) microscopy
of consortia from cytoline spiked with fatty acids was
studied (Fig. 2). Abundant globular bacteria, likely to
be fatty acid-oxidizing bacteria, were surrounded by a
core of long Methanosaeta-like methanogens intertwined with Methanospirillum-like cells. The presence
of fatty acid oxidizers along with Methanospirillum
spp. is well understood for their roles of H2/acetate producer and H2 scavenger.

Sequence analysis of archaeal 16S rRNA indicated


methanogenic assemblages from cytoline cluster as distinct clades close to known Methanospirillum sp. (clones
CLP-2 and CLB-2; 8992% similar) and Methanosaeta
(clone CLP-3 and CLB-3, 8794% similar) (Fig. 4).
These archaea were most abundant in cytoline, and to
a lesser degree in some of the other matrices as determined by microscopic analysis (data not shown), suggesting that cytoline favored the development of
aggregated juxtaposed communities of acetoclastic and
hydrogenotrophic methanogens with syntrophic
bacteria.

Bacterial phylogenetic analysis


Optimization of support matrix concentration
Consortia adhering to cytoline were characterized by
16S rRNA gene sequence analysis. Dominant RFLPs
from the high-CH4 producing cytoline matrix clustered
with Syntrophomonas sp., and Syntrophobacter sp.,
which are known butyrate and propionate oxidizing
bacteria (Fig. 3). However, sequences from propionate
and butyrate microcosms were 88% (clone CLP-1) and
90% similar (clone CLB-1) to 16S rRNA of sequences

Selected support matrices found to yield higher methanogenesis were evaluated over a range of dierent concentrations (data not shown). For propionate-induced
methanogenesis, cytoline, cytopore (2, 6, and 8 g/
25 ml), and cultispher (10, 250, and 500 mg/25 ml) were
evaluated. Methanogenesis from butyrate using montmorillonite (25, 250, and 500 mg/25 ml), cytoline (2, 6,

Fig. 2. Dierential interference contrast (DIC) photomicrographs of crushed cytoline after 4 weeks of incubation with butyrate. Fatty acid-oxidizing
bacteria in the core of the matrix are juxtaposed with Methanosaeta-like cells. Note the mesh-like structures formed by Methanosaeta-like
methanogens, which provide support to the fatty acid utilizing bacteria to reduce interbacterial contact for ecient transfer of reducing equivalents.

A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

889

Fig. 3. Phylogenetic tree of partial 16S rDNA sequences from domain Bacteria. Based on DNA isolated from crushed cytoline after 4 weeks of
incubation with fatty acids (propionate and butyrate). Numbers at nodes represent bootstrap values (100 times resampling analysis); only values that
are >50 are presented. Arthrobacter globiformis was used as outgroup.

and 8 g/25 ml), and cytodex (400 mg, 2 and 5 g/25 ml)
was evaluated. Methane was analyzed over time from
a range of support matrices to which consortia were
immobilized/entrapped and similar to previous experiments, cytoline at 8% (2 g/25 ml) yielded maximum
CH4 in minimum time period which was within 10 days
as compared to other matrices, indicating that increasing the amount of support matrices had no signicant
eect on their abilities to adhere to the CH4 forming

consortia. A recent study using concentrations of zeolite


in anaerobic digestion of piggery waste reported an increase of CH4 concentrations over a range of 24 g/L,
but methanogenesis declined with concentrations higher
than 4 g/L [15]. No such decline of CH4 at higher concentrations of cytoline tested (10% and 20%; data not
shown) was observed in our study, however, nor were
higher amounts of cytoline observed to stimulate increased methanogenesis over a period of weeks.

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A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

Fig. 4. Phylogenetic tree of partial 16S rDNA sequences from domain Archaea. Based on DNA isolated from crushed cytoline after 4 weeks of
incubation with fatty acids (propionate and butyrate). Numbers at nodes represent bootstrap values (100 times resampling); only values that are >50
are presented. Halobacterium halobium was used as outgroup.

Biomass estimation of consortia in selected support


matrices

also harbored correspondingly higher total protein


concentrations.

Estimation of biomass in high-CH4 compared to lowCH4 producing matrices was determined by total protein estimation (Figs. 5AF). The matrices that yielded
higher CH4, such as cytoline, montmorillonite, and the
hydrophobic membranes (teon and polypropylene),

Assessing consortium eciency against aerial


perturbations
The nal goal of this study was to evaluate the use of
these matrices to protect the adhering consortia from

A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

891

Fig. 5. Weekly total protein (biomass) estimation from sacriced support matrices spiked with propionate (P) or butyrate (B). (A,B) Bentonite,
montmorillonite, glass beads (106 and 425 to 600 lm), and BioSep beads. (C,D) Microcarriers exhibiting a range of surface characteristics and
porosities, including cytopore, cytoline, cultispher, and cytodex. (E,F) Hydrophobic (teon and polypropylene) and hydrophilic (nylon and
polysulfone) membranes.

exposure to air. Immobilized consortia such as those described in this study may serve, as inocula, which can be
conveniently added to a reactor in jeopardy, provided
strict anaerobic conditions need not be maintained dur-

ing re-inoculation. Inhibition of methanogenesis in a


failed bioreactor may be due to a chain of interlinked
events, including organic overloading (fatty acids and
acetate), low pH, and suciently high H2 levels causing

Fig. 6. Eect of exposure to air on immobilized syntrophicmethanogenic consortium shown with selected matrices. CH4 in the headspace was
estimated weekly to test the protective nature of matrices. (A) With propionate; and (B) with butyrate.

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A. Chauhan, A. Ogram / Biochemical and Biophysical Research Communications 327 (2005) 884893

the reactions to become endergonic. These cumulative


factors can cause the system to operate at a lower level
of eciency [6].
Consortia adhered to matrices exposed to air were
inoculated into fresh anaerobic medium, and methanogenesis was most rapid from cytoline-bound consortia.
Cytoline consists of an inert matrix of polyethylene,
with a hydrophobic surface, plus silica, which increases
the density of the beads and improves their characteristics for use in uidized bioreactors. This combination of
characteristics provides anchorage for cells and protection from shearing forces Fig. 6.
The results obtained from this study suggest the use
of support matrices as inocula that can be conveniently
added to a malfunctioning bioreactor. It is likely that
anaerobic consortia pre-grown in these matrices and
stored frozen may be introduced into a bioreactor. Since
the consortia will be present in the porous matrix, they
will quickly grow and restore the thermodynamics of
perturbed anaerobic digestor with minimal lag.

Acknowledgments
We thank Dr. Kerry Sublette, Tulsa University, for
providing BioSep beads, and Mr. Daniel T. Eyde,
GSA resources Inc., for montmorillonite as a gift. Funding for this study was provided by NASA space biotechnology Grant NAG-10-316. We are grateful to Ms.
Kristen Riley for making it possible for us to le a
timely patent.

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