Journal of Environmental Sciences 19(2007) 546–552
Alga-lysing bioreactor and dominant bacteria strain
PEI Hai-yan1 , HU Wen-rong2,3,∗, MU Rui-min3 , LI Xiao-cai3
1. College of Environmental Science and Engineering, Ocean University of China, Qingdao 266003, China. E-mail: [email protected]
2. Shandong Provincial Engineering Center on Environmental Science and Technology, Jinan 250061, China
3. School of Environmental Science and Engineering, Shandong University, Jinan 250061, China
Received 30 May 2006; revised 7 August 2006; accepted 10 October 2006
Abstract
Alga-lysing bacteria have been paid much attention to in recent years. In this study, the alga-lysing strain P05 which was isolated
from an immobilizing biosystem was immobilized by coke and elastic filler, forming two biological reactors. The removal efficiencies
of algae, NH4 + -N and organic matter using the two reactors were studied. The results showed that strain P05 was an ideal algal-lysing
bacteria strain because it was easy to be immobilized by coke and elastic filler which are of cheap, low biodegradability and the simple
immobilization procedure. After 7 d filming, the biological film could be formed and the reactors were used to treat the eutrophic water.
These two reactors were of stability and high effect with low cost and easy operation. The optimal hydraulic retention time of each
reactor was 4 h. The algae removal rates were 80.38% and 82.1% (in term of Chl-a) of coke reactor and filler reactor, respectively.
And that of NH4 + -N were 52.3% and 52.7%. The removal rates of CODMn were 39.03% and 39.64%. The strain P05 was identified as
Bacillus sp. by PCR amplification of the 16S rRNA gene, BLAST analysis, and comparison with sequences in the GenBank nucleotide
database.
Key words: alga-lysing; algae removal; immobilization; 16S rRNA; Bacillus sp.
Introduction
Many kinds of algae can lead to water bloom and red
tide in the seas, lakes and reservoirs. This phenomenon
influences or changes the physical and chemical character
of water and then results in many troubles in the producing
process of drinking water (Hargensheimer and Watson,
1996; Graham et al., 1998; MD et al., 2001), for example,
over-multiplication of algae can jam filter tanks, shorten
filter tank operating cycles, increase the back-flushing frequency, and raise the back-flushing water consumption and
running cost. Moreover, algae and their soluble metabolic products are the predecessors of by-products, which
are carcinogenic, teratogenic and mutagenic, produced
from chlorinating disinfections (Jeanine and James, 1998;
Wang, 2000).
Several researchers considered that the vanishing of
water bloom and red tide could be related to the infection
of the alga-lysing bacteria (Lee et al., 2000; Imamura et al.,
2001; Wu et al., 2002). As the biology of preventing and
curing of water bloom and red tide, the alga-lysing bacteria
have been concerned by more and more researchers. In
recent years, some alga-lysing bacteria have been reported
(Fukami et al., 1992; Imai et al., 1991, 1993, 1995;
Mitsutani et al., 1992; Sakata, 1990; Junichi et al., 1998).
However, most reports were limited to describe alga-lysing
phenomenon, isolation and identification by physiological
and biochemical methods. The application of alga-lysing
bacteria to remove algae in eutrophic water has not been
involved.
The removal of algae and microcystins through a biosystem immobilized on a sponge has been studied in our
research group. The results showed that when the hydraulic
retention time (HRT) was 5 h, the removal rates of algae
and microcystins were 90% and 94.17%, respectively, and
there were large numbers of bacilliform bacteria in this
biosystem (Pei and Hu, 2006).
In this study, strain P05, which has been isolated from
the biosystem, was applied to treat the eutrophic water by
immobilizing it in two reactors in which coke and elastic
filler have been filled as its carrier, respectively. The effects
of the strain P05 on removing algae, NH4 + -N and organic
matter in eutrophic water have been studied, and the strain
P05 has been identified by the sequence analysis of 16S
rDNA.
1 Materials and methods
1.1 Materials
Project supported by the Special Funds for Ph. D Research Station of
University (No. 20020422045) and the Science Foundation of Shandong
Province (No. Z2003B01) and the Environmental Protection Bureau of
Shandong Province, China. *Corresponding author.
E-mail: [email protected].
1.1.1 Strain P05
After isolated, cultured at 190 r/min and 30°C for 2 d,
P05 bacteria were used to form biological films in two
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Alga-lysing bioreactor and dominant bacteria strain
reactors with different kinds of filtering materials. The
concentration of strain P05 was (5–7)×107 cells/ml.
1.1.2 Bacterial culture medium
The Luria-Bertani (LB) bacterial medium contained
(g/100 ml): peptone 1, yeast extract 0.5, NaCl 10 (pH=7.0,
2% agar).
1.1.3 Water samples
The water samples were taken from a man-made lake
in Jinan City, China. The lake, fed by sewage has been
eutrophic. The main algae species in the samples included
Chlorella, Pediastrum, Ulothrix, Scenedesmus, Selenastrum of the Chlorophyta and Bacillariophyta, Navicula
of the Bacillariophyta and Microcystis aeruginosa of the
Cyanophyt. Chlorella was the dominant species, accounting for approximately 60% of the total algae in number.
The algae density was (0.48–1.32)×105 cells/L. The concentration of the chlorophyll-a (Chl-a) was 105.36–170.15
µg/L. The concentrations of NH4 + -N and CODMn were in
the range of 0.6–1.0 mg/L and 7.02–9.42 mg/L, respectively (Table 1).
1.1.4 Immobilization materials
Coke and elastic filler were chosen to immobilize strain
P05. The coke was collected from Coke Plant of Jinan
Steel and Iron Company, China. The elastic filler was provided by the National Institute for Environmental Studies,
Japan.
1.2 Isolation of alga-lysing bacteria
The alga-lysing bacteria were isolated from a biosystem
immobilized on a sponge that had a good removal effect on
algae and microcystins (Pei and Hu, 2006). Firstly, some
pieces of sponge were taken from the above biosystem
with sterile tweezers and placed into a 150-ml beaker with
20 ml axenic water. It was then broken with an electromagnetic stirrer under sterile conditions, namely initial
bacteria liquid. Secondly, mixing 1 ml the initial bacteria
liquid with 9 ml axenic water to obtain a 10−1 diluent.
Thirdly, mixing 1 ml 10−1 diluent with 9 ml axenic water
to get 10−2 diluent, the 10−3 , 10−4 , 10−5 diluents were
obtained as the above. Then, isolation and purification of
bacterial were carried out according to the reference (Shen
et al., 2002). Eighteen different bacteria strains have been
obtained. The 18 different bacteria strains with the same
concentration and volume were inoculated into the water
samples with 30% Chlorella, 30% Scenedesmus and 40%
M. aeruginosa, respectively. After culturing for 4 d at 190
r/min and 30°C, the algae removal effects were calculated
microscopically. Five bacteria strains with more than 75%
alga-lysing effect were obtained, and one of them named
P05 and studied in detail in this study.
547
1.3 Operating process
The operating process of reactor is shown in Fig.1, the
sizes of major parts of the reactor and working parameters
are shown in Table 2.
Fig. 1 Schematic diagram of coke or elastic filler reactor. (1) water tank;
(2) flowmeter; (3) influent pipe; (4) effluent pipe; (5) reactor; (6) air pump;
(7) valve; (8) gas flowmeter; (9) aerator; (10) coke (particle size was 2–3
cm); (11) coke (particle size was 1–2 cm); (12) coke (particle size was
0.5–1 cm); (13) water pump; (14) valve; (15) flowmeter. In the reactor of
elastic filler, 10–12 was full of filler.
Table 2 Sizes of major parts of the reactor and working parameters
Parameter
Value
Height of reactor (m)
Diameter of reactor (m)
Height of filler (m)
Particle size of coke (cm)
Back-flushing frequency (week-1 )
1.5
5.5
0.75
0.5–3.0
1.0
The cultures of P05 strain were poured into the reactors
respectively. Immobilization of P05 was carried on by
cycling bacteria culture mixed with the water sample,
referred to the reference (Li et al., 2002).
After immobilization, water sample was put in the
reactors and aerated continually. The treatment effects of
each reactor were measured under the condition that the
hydraulic retention times were 2, 3, 4, and 5 h, respectively.
1.4 Identification of Gram stain and 16S rRNA sequence analysis
Gram stain was refered to reference (Shen et al., 2002).
1.4.1 Bacteria cultivation and preparation of total genomic DNA
The strain was cultivated on potato extract and Bennett’s
culture medium at 30°C for 48 h, and then the cells
were harvested by centrifugation. After the cells were
washed twice with sterile water and TE buffer, and 40
µl TE buffer containing 10 mg/ml lysozyme was added,
and maintained at 37°C overnight. Standard procedures of
Pitcher (Preheim et al., 1991) were used for preparation of
Table 1 Parameters of water samples
Parameter
Color
Turbidity (NTU)
pH
Temperature (°C)
Chl-a (µg/L)
Algae density (×105 cells/ml)
Value
Dark green
24–30
6.92
20–29
105.36–170.15
0.48–1.32
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PEI Hai-yan et al.
total DNA from the strains. The DNA was maintained at
4°C after detecting the purity and concentration.
1.4.2 PCR primers and probe sequences
16S rRNA sequence was amplified from total genomic DNA by PCR with two general bacterial primers
(27F: 50 -GAGAGTTTGATCCTGGCTCAG-3, 1495R: 50 CTACGGCTACC TTGTTACGA-30 ). The products were
directly sequenced by the Taq DyeDeoxy Terminator
Cycle Sequencing Kit. The electrophoresis and data collection were made automatically by an Applied Biosystems
DNA Sequencer (model 377).
1.4.3 Phylogenetic analyses
16S rRNA sequences used for construction of the phylogenetic tree were retrieved from GenBank. The names
of the bacteria and the accession numbers are listed in
Table 3. P01, P03 and P07 were three other alga-lysing
strains isolated by our research group. The phylogenetic
tree was calculated with the neighbor-joining algorithm
by the program NEIGHBOR of the PHYLIPP (version
3.6 software). Evolution distances were calculated by the
DNADIST. To check the consistency of the resulting
Table 3 Names of isolates and related bacteria strains and GenBank
accession numbers of sequences used for the construction of the
un-rooted phylogenetic tree
Bacteria name
Number of
strains
Accession number
in GenBank
Amphibacillus fermentum
Anoxybacillus kestanbolinensis
Bacillus sp.
Bacillus badius
B. caldovelox
Bacillus cereus
Bacillus humi
Bacillus soli
Bacillus vietnamensis
Bacillus pseudofirmus
Bacterium
Bacterium
Bacillus megaterium
Bacillus cereus biovar toyoi
Bacillus cereus
Bacillus thuringiensis
Bacillus fusiformis
Bacillus fusiformis
Bacillus silvestris
Bacillus anthracis
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
–
K1
KMM 3737
–
–
BCE310100
LMG 22168
LMG 21840
–
124-1
47083
JL-74
BME550462
BCE310100
AH 527
2000032755
–
DSM 2898T
SAFN-010
2000031671
P01#
P03#
P05#
P07#
AF418603
AY248709
AY228462
AB098575
Z26925
AJ310100
AJ627209
AJ542515
AB099708
AB201799
AF227837
AY745842
AJ550462
AJ310100
AF290555
AY138282
AY472114
AJ310083
AY167818
AY138370
AY964602
AY964603
AY822613
AY822614
#
Isolated alga-lysing bacteria strain by our research group.
Vol. 19
tree, random resampling of the sequences (bootstrapping)
was performed, and a tree representing a consensus of
100 trees was obtained. Similarities were calculated from
partial sequences by considering all available overlapping
regions, with the exclusion of ambiguous nucleotides.
1.5 Analytical methods and apparatus
The changes of Chl-a concentration and algal density
between influent and effluent water were used to assess
the algal removal rate. The measurements of Chl-a, NH4 + N and CODMn concentration were referred to standard
methods (EPAC, 2002). The algae were counted with the
XSP-Microscope. The biofilm was observed by a microscope (XSP, Shanghai Analytical Apparatus Manufactory,
China) and a scan electron microscope (Jeol JSM T-300,
Hitachi, Japan).
2 Results and discussion
2.1 Biofilm forming
During the process of filming, the removal rate of Chla was measured and the change of the biofilm on the
immobilization materials was observed. The results are
shown in Fig.2 and Table 4. On the first day of filming,
these two reactors had removal effect on Chl-a, and there
was almost no biofilm on the coke or elastic filler at
that time, which show that these two materials have good
adsorption or filtration on algae. The Chl-a removal rate
of coke reactor was 62.9% and that of elastic filler reactor
was 29.8%, indicating the adsorption and filtration of coke
was better than that of elastic filler. On day 2, there were
shallow filemot, dotted film forming on the coke and elastic
filler, but the quantity was small. The removal rate of
coke reactor decreased to 52.5%, accounting for that the
adsorption of coke to algae attained saturation and the
biodegradation of biofilm was little at that time. From day
4, lumpish film was found on coke and elastic filler and the
quantity was much more from day 5, connecting to a whole
on some part of the material. The removal rates of Chl-a of
these two reactors were appdroximately 75%. The results
show that the biofilm has matured after 7 d of filming and
the immobilization process finished.
2.2 Removal effects of algae
The removal rates of Chl-a and algae cell at different
HRT are shown in Fig.3.
The Chl-a concentration of the influent was from 335.7
to 373.2 µg/L. When HRT was 2 h, the Chl-a decreased
to 247.0 and 231.7 µg/L in the effluent of coke and
Table 4 Change of biofilm during the process of filming
Filming time (d)
Apparent shape
Colour
Thickness
Quantity
1
2
3−4
5
No biofilm clearly
Dotted film
Dotted and lumpish film
Lumplish, connecting to a whole
on some part of filler
Matured film
–
Very shallow filemot
Shallow filemot
Filemot
–
–
0.02–0.1 mm
0.02–0.2 mm
–∼+
+
++
+++
Dark brown
0.04–1 mm
++++
6–7
+: Small quantity; ++: high quantity; +++: higher quantity; ++++: biofilm was full of the material.
No. 5
Alga-lysing bioreactor and dominant bacteria strain
549
Fig. 2 Chl-a removal rate during the process of filming.
elastic filer reactor and the removal rates were 29.0% and
33.4%, respectively. The colour of the effluent water was
laurel-green and the smell was fishy odour, because high
concentration algae remained in effluent. The removal rate
increased as the HRT was prolonged. When the HRT was 4
h, the removal rates by the two reactors reached higher than
80% and there was no color or odour in outfall water. When
HRT prolonged to 5 h, the increasing of Chl-a removal rate
was only slight. It showed that the optimist HRT was 4 h.
The removal effect of algae cells was coincident with that
of Chl-a.
2.3 Removal of NH4 + -N and CODMn
NH4 + -N is a major parameter which can indicate the
eutrophication degree of water. In this test, the change of
NH4 + -N concentration of water samples before and after
treatment was used to assess the removal effects of NH4 + N by the two reactors. The results are shown in Fig.4.
The removal rates of NH4 + -N increased with prolonging
HRT. When HRT was 4 h, the concentration of NH4 + -N
decreased to 0.515 and 0.468 mg/L in the effluent of coke
and elastic filler reactor from 1.395 mg/L. The removal
rates by the two reactors were over 50%. When the HRT
prolonged from 4 to 5 h, the removal rates increased
Fig. 3 Algal removal effect at different HRT.
Fig. 4 NH4 + -N and CODMn removal effects at different HRT.
slightly.
The measurement of CODCr was conducted under the
condition that organic matter in algae cell is strongly
oxidized, so the content of organic matter measured with
potassium dichromate oxidation method includes the other
organic matter in the water and the algae. The change of
CODCr can not clearly show the change of organic matter
without algae (Wu et al., 1987). In order to show the
removal effect of organic matter except of algea by CODMn
was measured in this test in stead of CODCr .
The removal rates of CODMn at different HRT are shown
in Fig.4. The concentration of CODMn in influent was from
8.06 to 8.59 mg/L. The removal rates of CODMn increased
with prolonging HRT. When HRT was 4 h, the CODMn
concentration in the effluent were 5.19 and 5.29 mg/L and
the removal rates of CODMn were 39.03% and 39.64%, in
term of coke reactor and filler reactor, respectively. The
removal rates increased slightly with HRT prolonging from
4 to 5 h.
Biodegradation, bio-flocculation, adsorption, detachment and sedimentation are the major ways of pollutants
removal by biofilm (Wu and Wang, 2001; Hamid et al.,
2006; Ilgi and Fikret, 2002; Gary and Joann, 1997).
Pollutant removal by these two reactors also followed these
rules. During the test, the biofilm formed by the dominant
bacteria and the sedimentation sludge was observed. There
were amounts of living algae and algal shells on the surface
of biofilm and inside. This phenomena show that the bioflocculation and the adsorption of biofilm on algae reduced
the amount of algae in water, which was the first step of
algae removal in these two reactors. The existence of algae
shells in the biofilm spreading on or desquamating from
coke or elastic filler accounted for the biodegradation of
biofilm on algae.
550
PEI Hai-yan et al.
2.4 Stability of the two reactors removing pollutants
In order to test the stability of the two reactors removing
Chl-a, algal cells, NH4 + -N and CODMn , the two reactors were operated continuously for 4 weeks under the
condition that the HRT was 4 h, and the removal rates
were measured every day. The results are shown in Figs.5
and 6. The results showed that the two reactors were of
stability removing algae, NH4 + -N and CODMn . Although
their removal rates had some change with different influent,
they were kept at a high level. The removal rates of Chla by coke reactor were from 76.4%–85.4%. The NH4 + -N
removal rates were over 50%, and the highest was 70.29%.
CODMn removal rates were over 32.8%. Fig.6 shows that
the lowest removal rate of Chl-a was 74.0% by the elastic
filler reactor, the highest 91.2%. The removal rates of algal
cells were from 73.3% to 87.3%. The removal rates of
NH4 + -N and CODMn were from 52.7%–75.2% and 30%–
45%, respectively.
The results showed that strain P05 was an ideal algallysing bacteria strain. It is easy for P05 to be immobilized
Vol. 19
by coke and filler which are of cheap, low biodegradability
and the simple immobilization procedure.
2.5 Characters and the alge-lysing mechnism of strain
P05
The result of Gram stain is shown in Fig.7. The strain
P05 was Gram negative. It was pillar cell with ellipse
spore, sporulation, mobile and the size was 0.6 × 4.0 µm
to 1.0 × 4.0 µm.
Algae lysis by bacteria may be brought about by the
production of extracellular products (Fukami et al., 1992;
Imai et al., 1995) or cell-to-cell contact mechanisms (Imai
et al., 1991, 1993; Mitsutani et al., 1992; Sakata, 1990).
The alga-lysing mechanism of strain P05 was studied
using the same method as that of strain P07 (Pei et al.,
2005a) and strain P15 (Pei et al., 2005b). The results
suggested that the lytic effect on algae of strain P05 was
performed by the production of extracellular products. The
extracellular products lysed the cell wall of algae firstly
and then degradated the Chl-a. So, it could be seen that
Fig. 5 Removal effects of pollutants by coke reactor in 4 weeks.
Fig. 6 Removal effects of pollutants by elastic filler reactor in 4 weeks.
No. 5
Alga-lysing bioreactor and dominant bacteria strain
551
(Mitsutani et al., 1992) and Myxobacter sp. (Junichi et al.,
1998). Bacillus was a new genus reported to have an algalysing effect.
3 Conclusions
Fig. 7 Pattern of strain P05 under the microscopy.
the removal rate in term of algal concentration was slightly
greater than that of according to Chl-a concentration at the
same HRT, from Fig.3.
2.6 Strain identification with 16S rDNA sequence analysis
The length of the PCR product of strain P05 was
1.5 kb. The sequences obtained in this study have been
deposited in the GenBank database under accession number AY822613. The DNA sequence similarity searches
showed that strain P05 shared more than 99.7% sequence
homology with certain strains of Bacillus. The sequence
homologies of P05 and four Bacillus fusiformis were
99.86% and 99.79%. According to these results, strain
P05 belonged to Bacillus in the phylogenetic framework
of bacterial classification. The phylogenetic tree is shown
in Fig.8.
Those reported alga-lysing bacteria included Alteromonas sp. (Imai et al., 1995), Flavobacterium sp.
(Fukami et al., 1992), Cytophaga sp. (Imai et al., 1991,
1993; Mitsutani et al., 1992; Sakata, 1990), Saprospira sp.
Fig. 8 Phylogenetic tree of strain P05. The scale bar indicates the number
of substitutions per sequence position.
The results showed that strain P05 was an ideal algallysing bacteria strain because it is easy to be immobilized
by coke and elastic filler which are of cheap, low
biodegradability and the simple immobilization procedure.
After 7 d filming, the biological film could be formed and
the reactors were used to treat the eutrophic water. These
two reactors were of stability and high effect, low cost and
easy operation.
The optimal HRT of each reactor was 4 h. The algae
removal rates were 80.38% and 82.1% (in term of Chla) of coke reactor and filler reactor, respectively. And that
of NH4 + -N were 52.3% and 52.7%. The removal rates of
CODMn were 39.03% and 39.64%.
Strain P05 was Gram negative, pillar cell with ellipse
spore, sporulation, mobile and cell size was 0.6 × 4.0
µm to 1.0 × 4.0 µm. Strain P05 shared more than 99.7%
sequence homology with certain strains of Bacillus. P05
belonged to Bacillus in the phylogenetic framework of
bacterial classification.
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