CHARACTERIZATION OF ANAEROBIC BIOMASS IMMOBILIZED IN POLYURETHANE FOAM

MATRICES FROM HAIS REACTOR BY SCANNING ELECTRON MICROSCOPY

Varesche M, Zaiat M, Vieira L, Vazoller R, Foresti E

Escola de Engenharia de São Carlos - Universidade de São Paulo

Av. Dr. Carlos Botelho, 1465, CEP: 13560-250, São Carlos, SP, Brasil

This paper reports on the characterization of immobilized anaerobic biomass by scanning electron
microscopy (SEM) using the hexamethyldesilazane (HMDS) technique. Polyurethane foam matrices
containing anaerobic sludge were taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor
treating glucose based substrate operating with bed porosities (ε) of 0.24 and 0.4. After three weeks of
operation with ε of 0.24, different kind of bacteria were found. Rods, coccus and Methanothrix-like
morphologies appeared into the support as small granules (micro-granules) or attached to the matrix as
disperse cells. Such micro-granules were attached to or entrapment in the matrices. Micro-granules with
409±15 µm in size were observed in the polyurethane foam matrices taken from HAIS reactor operating with
ε of 0.4. The bacterial morphologies were similar to those found in samples taken during the operation with ε
of 0.24, but acidogenic fat rods were predominant in the first HAIS reactor’s segment. The intense
acidogenic activity was verified by the excess of volatile fatty acids measured in this part of the reactor.
Microscopy observations permitted to conclude that anaerobic microrganisms were effectively immobilized
into the polyurethane foam support.

Palabras clave: anaerobic biomass, polyurethane foam matrix, scanning electron microscopy, HAIS reactor

INTRODUCTION

The increasing utilization of immobilized sludge for anaerobic wastewater treatment resulted in the
development of a great variety of reactor configurations. These units permit to obtain high cellular retention
times (CRT) even at low hydraulic detention times (HDT) since the biomass is properly attached to some
support material or self-immobilized as granules. Several cell immobilization supports have been used in
different reactors for anaerobic wastewater treatment, such as: agar gel (Karube et al., 1980; Del Nery,
1993), acrylamid (Inamori et al., 1989), porous ceramic (Zellner et al., 1987; Kawase et al., 1989),
polyurethane foam (Huysman et al., 1983; Fynn & Whitmore, 1984; Calzada et al., 1984; Gijgen et al.,
1988; Foresti et al., 1995). It is very important to know how the biomass attaches in the support to prevent
cell-wash-out from reactors, resulting in process failures. Huysman et al. (1983) tested a series of packing
materials to methanogenic bacteria immobilization and polyurethane foam appeared an excellent
colonization matrix. The authors concluded that the methanogenic biomass is mechanically retained in the
form of microcolonies within the porous material. In fact, Fynn & Whitmore (1984) observed that the
adhesion of methanogens to polyurethane foam is weak and so their removal from the support matrix is
relatively easy. Gijzen et al. (1988) verified the presence of aggregates of methanogenic bacteria in the
cavities of the polyurethane foam. However, these aggregates appeared to be physically associated with the
support material. Zaiat et al. (1996a) concluded that polyurethane foam matrices are completely adequate
for anaerobic sludge immobilization. For liquid superficial velocity as high as 2.21 cm/s applied to a fixed-
bed of immobilized anaerobic mixed culture in polyurethane foam, only 9% of the volatile suspended solids
(VSS) were washed-out from the matrices. This fact led to the hypothesis that mixed cultures of anaerobic
sludge attaches better in this type of support than a predominant methanogenic culture.

Aiming at the development of a new anaerobic reactor configuration, Zaiat et al. (1994) conceived the
horizontal-flow anaerobic immobilized sludge (HAIS) reactor utilizing polyurethane foam matrices for
biomass immobilization. Experiments carried out to evaluate the HAIS reactor performance proved its
efficiency in treating paper industry wastewater (Foresti et al., 1995)and synthetic glucose based substrate
(Zaiat et al., 1996b). In both the experiments, the start-up periods were very short (less than 10 days)and
polyurethane foam matrices were found to be adequate for anaerobic sludge immobilization. In fact, short
start-up periods of anaerobic reactors are only possible if the reactor provides a proper environment for
maintenance, growth, acclimatization and colonization of biomass within it. On the other hand, the behavior
of some monitoring performance parameters along the HAIS reactor’s length permitted to regard its flow
pattern as “ ideal plug-flow” . This fact also permits to infer on the importance of the reactor hydrodynamic
characteristics on the biomass dynamic within it.

This paper presents and discusses the results from scanning electron microscopy (SEM) analysis of
polyurethane foam matrices taken along the HAIS reactor’s length aiming to elucidate some aspects of
mixed-culture characteristics and distribution in fixed-bed anaerobic reactors.

MATERIAL AND METHODS

The polyurethane (PU) foam matrices with density of 20 Kg/m3 containing anaerobic sludge were taken from
a 2 liters horizontal flow anaerobic immobilized sludge (HAIS) reactor treating synthetic glucose based
substrate (Zaiat et al., 1996b). The bioparticles were obtained in four intermediate ports along the reactor’s
length as shown in Fig. 1.

Fig 1: Scheme of the horizontal-flow anaerobic immobilized sludge (HAIS) reactor.

Samples for scanning electron microscopy (SEM) were subjected to technique developed by Nation (1983)
and adapted to bacterial biofilms by Araújo (1994). Samples were firstly fixed in 0.1 M phosphate buffer (pH
= 7.3) containing 2.5 % glutaraldehyde for 12 hours at 4°C. The fixed samples were rinsed three times in 0.1
M phosphate buffer (pH = 7.3) for 10 min and dehydrated by using 50%, 70%, 80%, 90% and 95% ethanol
solutions during 10 min in each concentration. Each bioparticle was washed three times in 100% ethanol for
10 min and fixed by washing twice during 30 s with hexamethyldesilazane (HMDS). The samples were dried
at temperature lower than 60°C during 2 hours and coated with gold. SEM analyses were performed with a
Digital Scanning Microscope Zeiss DSM-960.

For optical microscopy observation, the sludge was detached from the bioparticles with distilled water.
Phase contrast observations were made with an Olympus microscope model BH2 and the
microphotographs were taken with an Exposure Control Unit Olympus.

RESULTS AND DISCUSSION

Prior tests were carried out to evaluate the effect of the HMDS technique on the physical structure of the
polyurethane foam (PU-foam). Fig. 2a shows SEM micrographs of the support material subjected to the
described technique and Fig. 2b presents the same material without such a treatment. No physical
modifications were observed in the support, permitting to adopt the described technique for analyzing the
bioparticles by SEM.

The anaerobic microrganisms in PU-foam was first evaluated by observations of bioparticle samples taken
from port 1 of the HAIS reactor operating with bed porosity of 0.24 and mean hydraulic detention time of 4.8
hours. Sampling occurred after the start-up period when a constant methane concentration in the off-gas
indicated that the HAIS reactor was being operated under operational stability. Three patterns of sludge
immobilization were observed by this analysis. The microrganisms were found to be immobilized as micro-
granules mechanically retained in the porous of the support (Fig. 3a), attached to PU-foam particles (Fig.
3b) or as disperse cells attached to the particle (Fig. 3c). The two last patterns indicate that the
microrganisms were firmly attached to PU-foam matrices probably by some kind of link. In fact, bacterial
adsorption depends on the electrical double-layer forces between the cell and the inert surface, London-van
der Waals forces and chemical bonding (hydrophobic and ionic bonds) being the predominant forces that
govern the attractive interaction between the surfaces (Sanchez et al., 1994). However, Huysman et al.
(1983) indicated that methanogenic biomass are probably not electrostatically adherent to foam matrix.
Therefore, it is possible that mixed cultures of anaerobic sludge attaches better in this type of support than
predominant methanogenic cultures. Different bacterial morphologies were observed in the support
material, irregular rods, coccus (Figs. 4a and 4b) and Methanotrix-like cells were observed in the
bioparticles (Fig. 4c).

Bioparticles from HAIS reactor operating with bed porosity of 0.4 and HDT of 8.0 hours were also observed
by SEM. Fig. 5 shows different characteristic sizes of the micro-granules immobilized present in the
sampling ports. A mean value of 409 ± 15 µm was observed for micro-granules obtained from the sampling
ports 1, 2 and 3 while samples from port 4 presented characteristic size of 268 µm. This difference in size
can be related to the hydrodynamic characteristic of the HAIS reactor. In fact, the ideal plug-flow regimen
provides spatial variation of substrate concentration resulting in different substrate/microrganisms ratio
along the reactor’s length. Table 1 shows values of chemical oxygen demand (COD) and volatile fatty acids
(VFA) in each sampling port of the HAIS reactor operating in steady-state. The low COD concentration
observed in the sampling port 4 makes the effective diffusion rate to be very low in this reactor’s segment.
Possibly, this was the main factor responsible for the lower micro-granule characteristic size in this region.

Table1: Values of chemical oxygen demand (COD) and volatile fatty acids (VFA) in each sampling port of
the HAIS reactor operating in steady-state.
Sampling port COD (mg/l) VFA (mg/l as HAc)
1 582 194
2 206 79
3 108 42
4 55 19

Observations of bioparticles obtained from HAIS reactor operating with ε of 0.4 by SEM detected the same
bacterial morphologies observed previously. In bioparticles taken from sampling port 1, it was observed the
predominance of non-methanogenic rods (Fig. 6a), also verified by phase contrast and fluorescence
microscopy (Fig. 6b). These anaerobic microbiota were probably responsible by the high COD removal
efficiency observed in the first section of the reactor (Table 1). In fact, the predominance of the acidogenic
activity in the first HAIS reactor’s segment is demonstrated by the excess of VFA generated in that region,
in spite of the presence of methanogenic rods (Fig. 6c) and Methanotrix-like bacteria (Figs. 6d and 6e)
which were also visualized.

The excess of VFA generated in the first section of the reactor and the remaining glucose were uptaken
gradually along the reactor’s length by the prevalent microbial anaerobic trophic groups immobilized in the
bioparticles (Figs. 6f, 6g and 6h).

As a general rule, the biomass immobilized as granular sludge obtained from up-flow anaerobic sludge
blanket (UASB) reactors has shown a well-structured conformation that optimizes substrate conversion and
metabolic products generation/ degradation. However, the spatial distribution of bacteria inside the
polyurethane foam matrices does not reveal any well-structured biomass conformation. The spatial
conformation of the microrganisms inside the support media clearly differs from those observed in granules
obtained from UASB reactors, where the biomass is well-structured in different layers (Guiot et al., 1991).
This unstructured spatial distribution can be an important factor to provide short periods of start-up resulting
in optimum utilization of primary substrates and intermediate products as observed by Zaiat et al. (1996b).

ACKNOWLEDGMENTS

This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). We
also thank to Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq), EDMIL Indústria e Comércio
for supplying the polyurethane foam and IFSC - Instituto de Física de São Carlos for the micrographic
analyses.

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 FIGURE CAPTIONS

Fig. 2: SEM micrographs showing the physical struture of PU-foam matrix subjected (a) and no-subjecte (b)
to HMDS technique.

Fig. 3: Three distinct types of sludge immobilization in PU-foam cubic matrices: (a) micro-granule
mechanically retained in the porous; (b) micro-granule attached to PU-foam particle; (c) disperse cells
attached to the particle.

Fig. 4: Microbial species observed in PU-foam matrices taken from port 1 of the HAIS reactor operating with
bed porosity of 0.24: (a) and (b) Irregular rods and coccus methanogenic and non-methanogenic; (c)
Methanotrix-like bacteria.

Fig. 5: Different characteristic sizes of the micro-granules immobilized in PU-foam matrices taken from
sampling ports 1 (a), 2 (b), 3 (c) and 4 (d) of the HAIS reactor with bed porosity of 0.4.

Fig. 6: Bacterial morphologies observed in the bioparticles obtained from HAIS reactor operating with ε of
0.4 by SEM. Bioparticles taken from sampling port 1: (a) non-methanogenic rods; (b) non-methanogenic
rods verified by phase contrast microscopy; (c) methanogenic rods; (d) Methanotrix-like bacteria; (e)
Methanotrix-like bacteria verified by phase contrast microscopy. Bioparticles taken from sampling ports 2, 3
and 4:(f), (g) and (h) anaerobic trophic groups immobilized in the bioparticles.