microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity pcr...

Water Research 37 (2003) 2259–2268

Microbial diversity in a thermophilic aerobic biofilm process:analysis by length heterogeneity PCR (LH-PCR)

Marja A. Tiirola1, Juhani E. Suvilampi, Markku S. Kulomaa, Jukka A. Rintala*

Department of Biological and Environmental Science, P.O. Box 35, Jyv .askyl .a FIN-40351, University of Jyv .askyl .a, Finland

Received 15 March 2001; received in revised form 4 November 2002; accepted 29 November 2002

Abstract

A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site

treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length

heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first

evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment

contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length.

The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier

elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging

to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most

prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations,

which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-

like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed,

the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study

shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater

processes.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Microbial diversity; Thermophilic; Aerobic; Biofilm process; Pulp and paper mill; LH-PCR

1. Introduction

Biological processes, both aerobic and anaerobic, are

widely applied in the treatment of pulp and paper

industry wastewaters. Although wastewaters are often

hot (about 50–60�C), biological processes are currently

run under mesophilic conditions, at temperatures below

40�C. Thus, many pulp and paper mill wastewaters are

cooled before being subjected to mesophilic treatment.

Biological treatment at high temperatures could be a

preferred option in the treatment of hot wastewaters and

in the internal treatment of circulating process waters,

since it would reduce operating and investment costs as

heat exchangers would not be needed and simpler

process configurations could be applied. However, it

has been reported that thermophilic bacteria may fail to

aggregate, making biomass separation from the treated

effluent a key design criterion. Currently, little is known

about the microbiological aspects of thermophilic

aerobic wastewater treatment processes. The weak floc

formation ability of these processes may be due to lack

of floc-forming species [1] or the porous nature and

pinpoint size of the flocs [2]. In previous microbial

cultivation studies, only Bacillus and Bacillus-like

organisms have been isolated from thermophilic aerobic

wastewater treatment reactors (for a review, see [1]). In

biofilm processes, such as the suspended carrier biofilm

process (SCBP), a biofilm is formed on the carrier

*Corresponding author.

E-mail address: [email protected] (J.A. Rintala).1Also for correspondence.

0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0043-1354(02)00631-0

surface preventing the degrading population from

washing out. Thermophilic aerobic biofilm processes

have so far been studied only in a few pilot or laboratory

studies. These studies have suggested stable perfor-

mance, high loading rates and short hydraulic retention

times (HRTs) [3,4,5,27].

Molecular techniques offer new possibilities for

profiling microbial dynamics which complement tradi-

tional cultivation studies. Most of these techniques are

based on the genes encoding ribosomal RNA (rDNA

genes). One of the recently introduced molecular

methods is length heterogeneity analysis of polymerase

chain reaction-amplified DNA (LH-PCR) of 16S rDNA

[6]. The analysis is based on the natural length variation

within 16S rDNA genes. The variable region is amplified

by PCR with fluorescently labeled universal primers that

recognize the region in all eubacteria. The peak

intensities within each LH-PCR size class are assumed

to be proportional to the original template concentra-

tions. The LH-PCR-method has been applied in screen-

ing microbial diversity in bacterioplankton [6] and, more

recently, in the examination of soil samples as well as the

origin of microbial faecal pollution in coastal waters

[7,8]. According to Ritchie et al. [8], LH-PCR is an easy,

rapid and reproducible method, but the authors

encountered several problem areas that require further

examination. First, the use of commercial fluorescent

size standards yielded fragment lengths that did not

correspond to the lengths of the sequenced 16S rDNA

fragments. Second, although more than 30 000 partial or

complete 16S rDNA sequences from cultivated or

uncultivated bacterial species have been submitted to

the public databases, there exists no exhaustive fragment

length database to directly compare and associate LH-

PCR lengths with native microorganisms. Comparison

of fragment lengths of native bacteria in different

phylogenetic groups is of especial importance, because

the usefulness of length heterogeneity analysis relies on

the evolutionary rate (deletions and insertions) of the

variable region of 16S rDNA that is selected for

analysis.

The objective of our study was to apply LH-PCR to

characterize bacterial populations in an on-site pilot

study using an SCBP to treat groundwood mill (GWM)

circulation water at temperatures between 44�C and

59�C. The process performance is described in greater

detail by Suvilampi et al. [9]. The microbial community

in both the suspended biomass and the biofilm attached

to carrier elements was analyzed from several samples

obtained during stable operation and during operational

disturbances. The population profiles were also com-

pared with those present in the feed and inoculum. A

computer-assisted analysis of the length variation of the

fragment sizes in different phylogenetic groups was

conducted to reveal the usefulness and limitations of the

LH-PCR method, and to assist in the interpretation of

the resulting LH-PCR patterns. For comparison, a

second dimension, GC-%, was also analyzed. GC-% is a

parameter that affects the mobility of DNA fragments in

denaturing gradient gel electrophoresis (DGGE) [10], a

commonly used molecular method for analyzing com-

plex microbial communities.

2. Materials and methods

2.1. Process water

The GWM circulation water used in all the experi-

ments was obtained from the grinder whitewater lining,

a stream also containing varying amounts of whitewater

from the paper machine. The characteristics of the

influent process water after 1mm pre-screening are

shown in Table 1.

2.2. SCBP pilot process and experimental set-up

Two serial aerobic reactors (referred to as R1 and R2

as single and R12 as serial reactors) with total volume of

2m3 were run in a 37-day trial. Half of the reactor

volume was filled with carrier elements (Flootek RF 438

black in R1 and RF 438 gray in R2, densities 0.95 and

1.05 gml�3, respectively), which provided an effective

surface area of 190m2m�3 in both reactors. A 250 l

lamell settling unit with sludge recirculation was

installed after the reactor stage. The technical operation

of the process was first tested by continuous feeding with

the whitewater for 4 days. Subsequently, on experi-

mental day 0, the reactors were inoculated with 60 l

(volatile suspended solids (VSS) 8.6 g l�1) of mixed

liquor from the mesophilic full-scale activated sludge

plant treating pulp and paper mill wastewater. The

inoculation temperature was 50�C.

2.3. Sampling and analyses

An automatic sampling device collected 24 h compo-

site samples from the feed line after screening, effluent

Table 1

Characteristics of groundwood mill circulation water used as

feed

N Average7SD

Temperature (�C) 81 6075

SS (mg l�1) 18 4607270

VSS (mg l�1) 18 4407260

DOC (mg l�1) 32 430790

TCOD (mg l�1) 9 13007260

SCOD (mg l�1) 9 11007250

SBOD7 (mg l�1) 9 6507140

N=number of samples, SD=standard deviation.

M.A. Tiirola et al. / Water Research 37 (2003) 2259–22682260

from R1, and final effluent after R2. Samples were

stored at �20�C if not immediately analyzed. VSS were

analyzed according to Standard Methods [11]. Scraped

biomass of the carriers was used to determine VS-fix.

Total VSS in the reactors was the sum of VSS and VS-

fix. Dissolved organic carbon (DOC) was measured with

a Shimadzu TOC-5050A total organic carbon analyzer.

Total and soluble chemical oxygen demand (TCOD and

SCOD) and soluble biological oxygen demand (SBOD7)

were analyzed according to Finnish standards [12,13],

respectively. All the samples were S&S GFA-filtered

except DOC and SCOD-samples were S&S GF50

filtered and TCOD samples were non-filtered.

2.4. Database examination

The 16S rDNA sequences, bases from 8 to 534

according to E. coli numbering [14], of 10 major lineages

of the bacteria domain were retrieved from the EMBL

database release 55 using the sequence retrieval system

(SRS) (EBI, Hinxton, UK). All the classified sequences

that were long enough were collected from the

phylogenetical groups of Cytophaga/Flexibacter/Bacter-

oides, epsilon-Proteobacteria, cyanobacteria, and Ther-

mus/Deinococcus. From the other groups the sequences

were limited to 150–200 per group by systematic

sampling to avoid overrepresentation of some groups.

For the Gram-positive group with low G+C content,

sequences were collected by the search words Clostri-

diaceae, Bacillaceae, Lactobacillaceae and Streptococca-

ceae. Sequences from uncultured species were generally

not classified and therefore could not be included. The

final data set comprised 1263 sequences. The GCG

program package (Genetics Computer Group, Madison,

WI) was used for the computational analysis of

fragment lengths and guanidine and cytosine content

(GC-%). The LH-PCR length data can be obtained

from M.T. on request.

2.5. DNA extraction

The biofilm of a plastic carrier was scraped mechani-

cally into 100ml of sterile water. 1ml sample of the

carrier biomass or suspended biomass was centrifuged in

a microcentrifuge tube at 14 000g 10min, and the pellet

was used for the analysis. The tube was filled with 0.4ml

of extraction buffer (10mM Tris–HCl pH 8.0, 1mM

EDTA, 0.2mgml�1 proteinase K, 1% sodium dodecyl

sulfate), and incubated at 37�C for 1 h. Cell lysis was

ensured by bead-millig: 0.6 g of glass beads (diameter

0.1mm) and 0.4ml of chloroform-isoamyl alcohol (24:1)

were added to the samples and the tubes were shaken at

1600 rpm for 3min using a cell homogenizer (Mikro-U

Dismembrator, B. Braun Biotech International, Mes-

slungen, Germany). The tubes were then centrifuged at

14 000g for 10min. The upper phase was re-extracted

with chloroform-isoamyl alcohol (25:24:1), purified with

ethanol precipitation (in the presence of 0.2M NaCl),

and finally dissolved into 100ml of molecular biology

grade water (50->30, Boulder, CO).

2.6. LH-PCR analysis

The primers for PCR were synthesized in the Institute

of Biotechnology at the University of Helsinki (Helsinki,

Finland) or in T–A–G–Copenhagen ApS (Copenhagen,

Denmark). Specific amplification of eubacterial

sequences was performed with primers fD1 (50-

AGAGTTTGATCCTGGCTCAG-30) [15] and an

IRD800 labeled PRUN518r (50-ATTACCGCGGCTG

CTGG-30) [10] for the LH-PCR analysis corresponding

to the fragment size of the computational analysis. For

the cloning and sequencing experiments eubacterial

specific primers fD1 and Com2-Ph (50-CCGTCAATT

CCTTTGAGTTT-30) [16] were also used to amplify a

longer sequence (B900 bp) of 16S rDNA gene. In the

PCR reactions, 1 ml of purified DNA was used as a

template in 50 ml PCR mixture containing 0.2mM of

dNTPs, 0.3mM of each primer, 1�DynaZyme reaction

buffer, 0.2mgml�1 BSA and 2 U DynaZyme F501-KL

polymerase (FinnZymes, Espoo, Finland). The PCR

procedure included an initial denaturation step at 95�C

for 5min and 30 cycles of amplification (94�C for 30 s,

55�C for 1min and 72�C for 3min). Gel electrophoresis

was performed with an automated LI-COR 4200

sequencer (LI-COR BioTech, Lincoln, NE) for six hours

or overnight using 6% Long Ranger denaturing poly-

acrylamide gel (FMC Bioproducts, Rockland, ME).

Data were analyzed using Quantity One software (Bio-

Rad Laboratories, Hercules, CA). For the development

of DNA length standards, templates from selected

strains of Sphingomonas sp., Yersinia sp. and Lactoba-

cillus sp. were amplified with the same 16S rDNA-

specific PCR primers as the examined samples. The

products were cloned into the pGEM-T vector (Prome-

ga, Madison, WI), and analyzed by bidirectional

sequencing. Size standards of 470, 527 and 553 bp were

prepared by amplifying these clones by the same method

and primers as used for the reactor samples.

2.7. Cloning and sequencing

The eubacterial 16S rDNA products obtained from

the reactor samples were excised from 0.8% agarose gel

and purified with Ultrafree-DA filter units (Millipore,

Bedford, MA, USA). The products were cloned into the

pGEM-T vector and sequenced using simultaneous

bidirectional cycle sequencing with a SequiTherm Excel

II DNA Sequencing Kit (Epicentre Technologies,

Madison, WI), sequencing primer pairs T7/SP6 or

PRUN518r/fD1 labeled with IRD800 and IRD700 dyes,

and the automated LI-COR 4200 sequencer. The

M.A. Tiirola et al. / Water Research 37 (2003) 2259–2268 2261

sequences (in average 400–600 bp long) have been

deposited in the EMBL database under accession

numbers AJ303332–AJ303341 and AJ298266–

AJ298277. The SCBP sequences are marked in the

database with A, S, D or F depending on the DNA

source (attached biomass, suspended biomass, attached

biomass in disturbed conditions and in feed water,

respectively). Sequences were compared with the data-

base with the BLAST program [17].

3. Results

3.1. Thermophilic process performance and biomass

retention

The temperature in the two-stage process ranged from

44�C to 59�C according to the feed water temperature

(range 48–68�C). In the two-stage process (R12) and

single reactor (R1), loading rates were gradually

increased by decreasing the HRT (Table 2). During the

run, R1 removed generally 47–55% and R12 55–70% of

DOC. The processes experienced four alkaline pH

shocks, which decreased only temporarily (generally

for 1 day) the DOC removal, especially in R1. On day 8

the DOC removal in R12 declined from 60% to 41%

after two pH shocks lasting about 10 h on days 7 and 8

(pH up to 12 in R1 and pH 9 in R2). DOC removal

recovered to 55% within 6 h following the last pH shock.

During the disturbances, the temperature was lower (40–

45�C) than under normal operation (52–55�) owing to

the recovery period of 6–12 h during which the flow of

feed to the reactor was suspended.

The concentration of attached biomass increased in

both reactors attained a maximum of 2.6 kg VS-fixm�3

in R1 on day 24. On day 30, the concentration of

attached biomass decreased to 1.4 kg VS-fixm�3 owing

to washout. This was apparently caused by two pH

shocks; pH rose to 11 on days 24 and 29 (for 12 and 6 h,

respectively). In R2 the concentration of attached

biomass increased to 2.9 kg VS-fixm�3 during the run

despite the disturbances. Suspended biomass concentra-

tions in both reactors ranged typically from 0.6 to 1.2 kg

VSSm�3. These figures mean that 60–80% of the total

biomass (VS-fix+VSS) was attached on the carriers.

3.2. Computational analysis of length variation in

LH-PCR

Ribosomal sequences from ten major lineages of the

bacteria domain were examined to assist the general

interpretation of LH-PCR profiles and to reveal the

usefulness of the method. The fragment chosen for

Table 2

Temperature, pH, HRT, loading rate, biomass and DOC removal in the two-stage SCBP treating groundwood mill circulation water in

the first stage reactor (R1) and second stage reactor (R2) on the days when microbial samples were taken for molecular analyses

Day Reactor unit T (�C) pH HRTa

(h)

Loading rate (kg

SCODm�3 d�1)

Suspended and

attached biomass

(kg VSSm�3/kg

VS-fixm�3)

VS-fix/total VSS

ratio (%)

DOC

removala

(%)

3 R1 50 7.0 4.1 8.5 1.0/1.4 58 55

R2 49 7.5 8.3 4.2 1.0/1.0 50 60

8 R1 48 12.0 4.0 6.1 0.7/1.6 70 nd.

R2 46 9.0 8.0 3.1 0.6/1.2 67 40

13 R1 50 6.5 2.5 9.8 1.0/2.2 69 49

R2 50 7.2 5.0 4.9 0.8/1.2 60 69

17 R1 52 7.0 3.3 6.5 0.6/2.6 81 60

R2 51 7.5 6.7 3.3 0.4/2.1 84 73

21 R1 53 7.0 3.3 8.8 0.9/2.6 75 51

R2 52 7.5 6.7 4.4 0.5/2.1 81 65

30 R1 50 11.0 1.9 9.0 1.2/1.4 54 26

R2 50 9.0 3.8 4.5 1.0/2.1 68 50

35 R1 55 7.0 1.6 19.0 0.5/1.2 71 43

R2 55 7.5 3.2 9.5 0.5/2.5 83 55

aR2 figures HRT and DOC removal after two-stage process.


analysis contained the first third of the 16S rRNA gene,

including hypervariable regions V1–V3.

The analysis revealed that the sequence length of

partial 16S rDNA varied between 465 and 563 bp, while

the GC-% varied between 44% and 63% (Fig. 1). The

differences in fragment lengths were large enough to

allow easy separation of the short 16S rDNA sequences

of alpha-Proteobacteria and cyanobacteria from the

longer sequences even on a conventional agarose gel,

whereas in denaturing sequencing gels even base pair

differences can be separated. Some phylogenetic groups

had relatively conserved fragment sizes, but in general

there was a great deal of overlap. There was hardly any

case in which length determination would have served to

correctly assign the fragments to their correct phyloge-

netic group. However, practically all the fragments over

544 bp belonged to low G+C Gram-positive bacteria.

Of the different phylogenetic groups in the study, the

sequence lengths of that group were the most hetero-

geneous, extending from 490 to 563 bp. The sequence

information is based on the sampling of the current

database, and as more sequences are retrieved from

novel organisms, a wider range of fragment lengths may

be observed.

3.3. Molecular analysis of the biomass in the treatment

process

Inoculum. Thermophilic SCBP was started using an

inoculum from a full-scale mesophilic activated sludge

plant treating wastewaters of integrated pulp and paper

mill and bleached kraft mill effluent. On the basis of the

LH-PCR analysis, short-length PCR products (468–

472 bp) dominated the inoculum (Fig. 2A), accounting

for up to 40% of the calculated total peak area.

Computational analysis of LH-PCR lengths in different

bacterial groups suggested that short rDNA templates

are derived either from cyanobacteria or from members

of alpha-Proteobacteria (Fig. 1). Because the wastewater

treatment process reduces organic compounds in the

waste stream, it is most likely that heterotrophic

organisms, such as alpha-Proteobacteria, were dominant

in the samples.

Feed water. The LH-PCR analysis from the feed water

of the pilot process showed a stable bacterial community

profile. The most prominent peaks represented lengths

518, 519 and 521 bp, and these peaks usually comprised

more than 90% of the total peak area (day 3 shown in

Fig. 2A). The computational analysis revealed that there

are several phylogenetic groups that are predicted to

yield fragments of about 520 bp.

Normal SCBP operation. During normal operation

(no pH shocks or aeration failures), the attached

biomass of the reactor R1 yielded PCR fragments of

many different size classes with ten or more LH-PCR

peaks, revealing the complexity of the microbial com-

munity (Fig. 2B). However, no bands representing

lengths 509–512 bp were observed thus excluding pre-

sence of the genus Thermus. Thermus sequences create a

compact group with lengths of about 509–512 bp

(Fig. 1). Band sizes 518 and, especially, 521 bp were

generally the most prominent size classes in both the

44

46

48

50

52

54

56

58

60

62

64

460 480 500 520 540 560

Length (bp)

GC

-con

tent

(%

)

Alpha-Proteobacteria

Beta-Proteobacteria

Gamma-Proteobacteria

Delta-Proteobacteria

Epsilon-Proteobacteria

Cytophaga/Flexibacter/BacteroidesCyanobacteria

Thermus/Deinococcus

Gram-positives, low G+C

Gram-positives, high G+C

Fig. 1. Computational analysis of length and GC-content of partial 16S rDNA (region 8534, E. coli numbering) in phylogenetical

groups. The data consist of 1263 sequences obtained from the EMBL database.


serial reactors. However, reactor R2 had a slightly

different bacterial population compared to reactor R1

(Fig. 2D).

Suspended biomass samples showed clearly different

LH-PCR patterns as compared to the attached biomass,

indicating a different bacterial composition. Suspended

biomass produced high amounts of relatively long LH-

PCR fragments, especially 536 and 538 bp (Fig. 2C),

suggesting that Bacillus-related species may be abun-

dant. We made a complete analysis of all the Bacillus

and Paenibacillus sequences in the database (368

sequences), and found that most of the sequences

belonging to these genera (over 90%) yield lengths

between 531 and 538 bp.

Disturbed SCBP operation. The LH-PCR analysis

showed that after the pH shocks (days 8 and 30) the

attached biomass also experienced a major shift towards

microbial populations that produced higher fragment

Fig. 2. Fragment analysis of partial 16S rDNA amplified from samples of inoculated mesophilic sludge and feed water (day 3) (A),

attached biomass (B) and suspended biomass (C) of the reactor R1 of the pilot-scale aerobic thermophilic SCBP during normal

operating conditions. Comparisons of samples from the serial reactors R1 and R2 are shown in lower figures, (D) attached biomass

samples, day 17, during normal operating conditions, (E) attached biomass samples, day 8, during disturbed operating conditions.


lengths in LH-PCR analysis. This was observed

especially in reactor R1, which was more severely

disturbed (Figs. 2D and E).

Some of the PCR products were cloned and a total of

22 clones were selected to reveal the bacterial diversity in

the thermophilic process (Table 3). Although the

number of sequenced clones was small, the results show

that several Gram-negative genera from beta-Proteo-

bacteria, gamma-Proteobacteria and Cytophaga–Flexi-

bacter–Bacteroides group can thrive in the thermophilic

process as a part of the attached biomass (biofilm). All

these clones shared the LH-PCR length of 521 bp.

Sequences of species belonging to Bacillaceae (Anox-

ybacillus, Thermobacillus and Paenibacillus having LH-

PCR lengths 535, 550 and 538 bp, respectively) were

sequenced from the suspended biomass but also from

the biofilm sample collected during disturbed conditions

6 h after alkaline shock.

4. Discussion

In this study, the microbial diversity in an aerobic

thermophilic SCBP was analyzed by LH-PCR. This

method could offer a rapid tool for characterization of

microbial communities in many kinds of process

industry. Thermophilic aerobic processes have pre-

viously been studied using laboratory cultivation proce-

dures. Recent studies by Konopka et al. [28] and LaPara

et al. [18–21] have shown the advantages of the modern

culture-independent DNA methods, including DGGE

and sequence analysis of 16S rDNA clones.

According to our findings the microbial community of

the thermophilic SCBP exhibited wide bacterial diversity

in temperatures ranging from 44�C to 59�C. Archae-

abacteria-specific PCR primers were also tested, but no

amplification product was obtained from inoculum or

subsequently from feed water or reactor samples (data

not shown), which suggests that eubacteria thrive in this

thermophilic aerobic process. Noteworthy changes in

the microbial diversity of the biofilm were connected to

the occurrences of alkaline shock conditions, which may

also occur in full-scale mill conditions. During normal

operation, several different LH-PCR band patterns were

obtained from attached biomass, but a single dominant

band was observed most of the time. However, the

sequencing studies showed that even this main band was

a mixture of sequences from members of several

bacterial groups (at least beta- and gamma-Proteobac-

teria, and Cytophaga–Flexibacter–Bacteroides group).

Suspended biomass samples showed a great variance

suggesting that different microbial populations grew in

bursts. This was most likely due to repetitive adjust-

ments in the HRT. In a previous study [18], it has been

shown that the dominant phylotypes of the thermophilic

bioreactors change as a function of HRT. Therefore the

shifts in the community structure in the suspended

biomass samples may be due to decreased HRT during

operation of the pilot system, which increased the

selective pressure for the high growth rate. The

Table 3

Phylogenetic affiliation and closest cultured organisms of the cloned 16S rDNA sequences collected from the thermophilic wastewater

treatment system

Group Accession no. Source Best match in EMBL database Identity (%)

Alpha-Proteobacteria AJ303339 S Erythrobacter sp. AS–45 95

AJ303341 F Rhodobacter gluconicum acc. AB077986 92

Beta-Proteobacteria AJ303332 A Hydrogenophaga intermedia S1 95

AJ303334 A Hydrogenophaga palleronii DSM 63 96

AJ303337 A Hydrogenophilus thermoluteolus TH–1 98

AJ298276 D Tepidimonas ignava SPS-1037T 98

Gamma-Proteobacteria AJ303333 A Sulfur-oxidizing bacterium OAII2 91

Cytophaga/Flexibacter/Bacteroides AJ303335, AJ303336 A Cytophaga sp. BD2–2 94

Gram-positives, low G+C AJ303338, AJ303340, AJ298277 S, D Paenibacillus granivorans A30 94–95

AJ298267, AJ298268 F Alicyclobacillus acidocaldarius DSM 446 97–98

AJ298269-AJ298272 D Anoxybacillus gonensis G2T 97–99

AJ298273-AJ298275 D Thermobacillus xylanilyticus XE 95–98

Thermus/Deinococcus AJ298266 F Fervidobacterium islandicum AW–1 94

F, feed water (day 13); A, attached biomass (reactor R1, day 21); S, suspended biomass (reactor R1, day 21); D, attached biomass

during disturbed conditions (reactor R1, day 8).


differences between the attached and suspended biomass

can be explained by the different growth rates and/or

adhesion behaviors of bacterial groups. It is known that

early colonizing organisms are usually gram-negative

bacteria, whereas gram-positive bacteria have only

rarely been recorded on surfaces in aquatic habitats

[22]. However, after disturbed operation (alkaline shock)

Bacillus–related sequences were obtained also from the

attached biofilm. This result suggests that fast-growing

Gram-positive bacteria can colonize carrier surfaces, but

other bacteria with higher affinity for the substrate may

outcompete them from the biofilm environment under

stable conditions. Many of the most alkaliphilic micro-

organisms known are mesophilic bacteria of the genus

Bacillus [23]. Highly resistant cell walls, the tendency to

sporulate and, most importantly, short generation time

may favor these bacteria over others during or following

extreme conditions. Their short generation time may

also work in their favor in suspended biomass, since

slow-growing bacteria may be washed out during short

HRTs (down to 1.6 h in the single reactor).

The SCBP pilot study was established with two serial

aerated reactors. Most of the DOC removal occurred in

the first reactor R1 from which suspended biomass was

continuously washed out to the reactor R2. However,

suspended biomass content was higher in R1 than in R2

(effluent samples) which might indicate rapid reduction

in biomass weight by maintenance and decay processes

in the latter reactor. During normal operating condi-

tions, both the serial reactors showed very similar LH-

PCR profiles of bacterial diversity in the suspended

biomass samples, which is logical considering the

continuous inoculation from R1 Following pH-shock,

bacterial composition shifted more in the first reactor

(R1), in which the pH change was more severe.

However, the bacterial community of the process

recovered quickly, as revealed by process parameters

and molecular markers. Fast recovery is in accordance

with the high growth rate of thermophiles [24].

The mesophilic sludge used as an inoculum consisted

of a different microbial composition, as concluded from

its different LH-PCR patterns when compared to either

suspended or attached biomass in the thermophilic

SCBP. On the other hand, some of the prominent peaks

obtained from the attached biomass were similar to

those from the feed water from the pulp and paper mill

whitewater lining. However, computational calculations

revealed that many genera, even different subdivisions,

produce similar LH-PCR lengths. Nevertheless, LH-

PCR was shown to be convenient for preliminary

screening and selecting the samples that were of interest

for further experiments. Compared to DGGE, which is a

commonly used molecular tool for profiling bacterial

diversity, LH-PCR gives more reproducible results and

numerical values. In LH-PCR, fragment sizes can be

compared to exact lengths derived from the database. In

DGGE, the predictions of the mobility of the DNA

fragments in the gel (melting behavior) are only a guide,

as shown in the work of van Orsouw et al. [25]. Some of

the previous studies have also reported a discrepancy in

the LH-PCR analysis when using commercial fluores-

cently labeled size standards [7,8]. We also encountered

the same problem when using the capillary electrophor-

esis system (ABI310) and GeneScan-500 TAMRA size

standard (Applied Biosystems, Foster City, CA, USA).

The problem was solved easily and cost-efficiently by

using ‘home-made’ size standards that originated from

previously cloned ribosomal fragments. Using these

standards, correct band sizes could be obtained in the

LH-PCR analysis, as confirmed by sequencing.

The basic idea of LH-PCR is to use fluorescently

labeled primers to allow fast and accurate detection with

an automated sequencer. This approach does not allow

direct sequencing analysis of the bands of interest,

because the bands can not be excised from the gel. Other

electrophoresis systems can also be used. We have tested

a manual sequencing system and capillary electrophor-

esis system for LH-PCR analysis and found that these

systems have some notable advantages. Capillary

electrophoresis allowed faster (analysis time less than

1 h) and simpler operation, and might thus be more

practical in the daily control of industrial bioprocesses.

With a manual sequencing system, the whole analysis

including silver staining may take as long as 1–2 days,

but the manual sequencing apparatus is relatively

inexpensive and the method allows the extraction of

the band of interest for sequencing.

Currently, LH-PCR may be one of the most attractive

methods available, if one is seeking a fast, reproducible

and predictable method for the preliminary screening of

microbial systems over time and space. It could become

a valuable tool in screening microbial dynamics in

environmental and biotechnological fields. For future

development, a two-dimensional analysis of both

sequence length and G+C—content would be interest-

ing for the analysis of complex microbial communities.

As shown in the computational analysis (Fig. 1), the

phylogenetic division of many bacteria could be better

estimated by their two-dimensional coordinates. Two-

dimensional electrophoresis has been used in genetic

mutation analyses [26] using non-denaturing conditions

in length analysis and DGGE for the analysis of the

second dimension. However, as it is technically not yet

possible to analyze the first dimension in two-dimen-

sional electrophoresis using denaturing conditions,

measurements may be too inaccurate to yield relevant

numerical data, new technical solutions are needed. The

number of bacterial sequences available for comparison

are also constantly increasing; all that is lacking is the

availability of user-friendly biocomputing programs to

allow real-time simulations of sequence-based biomar-

kers of the database.


5. Conclusions

1. Database analysis of 16S rDNA sequences revealed

that the length heterogeneity of the first third of the

16S rDNA spans at least 465–563 bp.

2. Some qualitative predictions of the microbial com-

munity in bioprocesses can be made directly from the

LH-PCR length profiles, but for more detailed

analysis, sequencing or other DNA analyses are

needed.

3. The aerobic thermophilic biofilm process showed

stable performance on mill premises with generally

70–55% DOC removal in the two-stage process

with HRTs of 8–3 h. The community structure

differed from that of the seed sludge, and recovered

rapidly from disturbances caused by extreme pH

variations.

4. Different LH-PCR profiles for suspended and

attached biomass show that different microbial

populations follow as a function of the applied

selection criterion.

5. Alkaline pH shock led to a microbial community

where thermophilic Bacillus-associated microbes

were present, whereas the same bacteria did not play

significant role in the attached biofilm during non-

disturbed operation.

Acknowledgements

This study was supported by the funding of the

Graduate School for Environmental Ecology,

Ecotoxicology and Ecotechnology (EEEE) at the Uni-

versity of Jyv.askyl.a. We thank Maarit Kivim.aki for

assistance in the computational analysis, Irene Helkala

for technical assistance in laboratory, and Mervi

Ahlroth for helpful comments during the writing

process.

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