Aquaculture 500 (2019) 477–484

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Ammonia nitrogen and nitrite removal by a heterotrophic Sphingomonas sp. T

strain LPN080 and its potential application in aquaculture
Long Yuna,b,1, Zonghe Yua,c,1, Yinyin Lia,b, Peng Luoa,c, , Xiao Jianga,c, Yushun Tiana,b,c,
⁎

Xiongqi Dinga,b,c
a
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Provincial Key Laboratory of Applied Marine Biology (LAMB), South China Sea
Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 501301, PR China
b
University of Chinese Academy of Sciences,Beijing 100049, China
c
South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510301, PR China

ARTICLE INFO ABSTRACT

Keywords: A safe, effective and simple method for ammonia nitrogen and nitrite removal in farming water has always been a
Sphingomonas goal of environmentally friendly farming models aimed at water saving and reducing the discharge of farming
Aquaculture wastes. In this study, it was shown that a bacterial strain LPN080 isolated and screened from a shrimp culture pond
Farming waste treatment can efficiently remove both ammonia nitrogen and nitrite. When supplemented with ammonia nitrogen and glucose
Ammonia nitrogen
in water samples, ammonia nitrogen (as the only nitrogen source) was reduced from an initial 8 mg/l to 0.3 mg/l in
Nitrite
48 h, and a removal rate of ammonia nitrogen of up to 96% was achieved. With the same addition of glucose in the
water samples, nitrite (as the only nitrogen source) reduced from an initial 5 mg/l to 0.8 mg/l, and the removal rate
was approximately 81% in 48 h. The LPN080 strain was identified as a member of Sphingomonas using 16S rDNA
sequencing. Sphingomonas sp. LPN080 could indirectly inhibit the growth of Vibrio spp. in water supplemented with
glucose and the inhibition was not caused by the production of antimicrobial substances. Sphingomonas sp. LPN080
showed high biosafety toward shrimp Litopenaeus vannamei. All these results demonstrated that Sphingomonas sp.
LPN080 has a potential economic value in aquaculture and even in wastewater treatment.

1. Introduction and the discharge of farming wastes (Zhou et al., 2009; Crab et al.,
2012; Martínez-Córdova et al., 2015).
Aquaculture plays a vital role in improving the living standards of Ammonia nitrogen and nitrite are two of the most important indexes
humans. In 2011, the per capita fish supply exceeded 19 kg and has in farming water management as their toxicity can directly impact the
been steadily growing since then, aquaculture accounted for 43% of the survival of aquatic animals (Lin and Chen, 2003; Xian et al., 2011) and
world's fish production, and the yield of the shrimp culture reached the productivity of aquaculture (Ebeling et al., 2006; Muthukrishnan
21% (FAO, 2014). Because of the continuous increase in demand for et al., 2012). Thus, maintaining low concentrations of ammonia ni-
aquaculture products and the promotion of substantial economic ben- trogen and nitrite in farming water is a key target for successful culture.
efits, intensive aquaculture has quickly expanded in developing coun- In aquaculture, there are two main sources of ammonia nitrogen, the
tries (Avnimelech, 2009). Meanwhile, environmental pollution and metabolic waste of breeding animals themselves and the decomposition
input costs have been increasing (Tovar et al., 2000). With the ever- of remains used for food by microorganisms (Gross et al., 2004). Nitrite
increasing consciousness of environmental protection, China has been mainly comes from the oxidation metabolism of ammonia and accu-
strengthening its measures to control aquaculture pollution, and mulates becasue of imbalances in nitrifying bacterial activity (Wang
aquaculture effluent treatment has gradually become a necessary step et al., 2004). In traditional shrimp farming models, changing water is a
in aquaculture. Microbial-based farming systems represent one of the direct way to maintain low concentrations of ammonia nitrogen and
most viable strategies to achieve a sustainable aquaculture as beneficial nitrite (Wang, 2003). However, this strategy not only results expanding
microbes can improve water quality and decrease disease occurrences large amounts of farming wastes and potentially causing water

Corresponding author CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Provincial Key Laboratory of Applied Marine Biology
⁎

(LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 501301, PR China.
E-mail address: [email protected] (P. Luo).
1
The two authors contributed equally to this work.

https://doi.org/10.1016/j.aquaculture.2018.10.054
Received 30 May 2018; Received in revised form 22 October 2018; Accepted 23 October 2018
Available online 25 October 2018
0044-8486/ © 2018 Elsevier B.V. All rights reserved.
L. Yun et al. Aquaculture 500 (2019) 477–484

eutrophication, but also leads to the dissemination of pathogens that 2.3. Molecular identification of the selected strain
can be a latent hazard for the outbreaks of aquatic diseases (Kautsky
et al., 2000; Haseeb, 2012). A recirculating aquaculture system (RAS) A 16S rDNA-based method was used to identify the strain used. The
featuring the adoption of some water-processing equipment is also a genomic DNA of the strain was extracted using a Bacterial Genomic
common approach for controlling water quality (Timmons et al., 2002); DNA Extraction Kit (TaKaRa, China) for PCR amplification of 16S rDNA
however the high cost of RASs limits their wide application in devel- with primers 63F and 1387R (Lane, 1991). The PCR product was di-
oping countries. Subsequently, biological flocs culture (BFC) for the rectly sequenced by Sangon Biotech Company (Shanghai, China). The
regulation of the water quality by microbes is considered to be one of obtained sequence was queried against the GenBank database using
environmentally friendly farming models as farming wastes are treated BLASTN. A phylogenetic tree was constructed using MEGA5 (Tamura
by beneficial microbes and then reutilized by animals (Avnimelech, et al., 2011) based on the sequence of the strain and related sequences
2009; Zhou et al., 2009; Krummenauer et al., 2016) using the neighbor-joining method.
Generally, ammonia nitrogen and nitrite in BFC systems and RASs
are removed through nitrification processes by ammonia oxidizing 2.4. The effect of the selected strain on ammonia nitrogen removal
bacteria and subsequent nitrite oxidizing bacteria (Brune et al., 2004;
Velusamy and Krishnani, 2013). The process of nitrification performed A total of 1 l of the filtered and sterilized seawater containing am-
by autophytic bacteria, which require an attachment surface to function monia nitrogen and carbon sources (SSAC) was prepared by adding
or propagate, is very slow, and extra equipments such as biofilters are (NH4)2SO4 to adjust the concentration of ammonia nitrogen to 8 mg/l
needed in RAS systems (Fdz-Polanco et al., 2000; Egashira and Sato, and by adding 1 g of glucose. The selected strain was cultured overnight
2007). In contrast, heterotrophic ammonia assimilation can effectively and the optical density of the bacterial cells was adjusted to
remove ammonia nitrogen and convert it into bacterial biomass, which OD600nm = 0.8 (approximately 4 × 108 cfu/ml). Then 1 ml of the di-
serves as an important source of feed protein. Therefore, the use of luted bacterial fluid was centrifuged and washed twice with sterilized
bacteria that possess heterotrophic ammonia assimilation ability can seawater. In the experimental group, 300 μl of the suspended cells was
reduce the production cost and improve the overall economic level in added to 150 ml of SSAC, whilst in control group, 300 μl of sterilized
aquaculture (Avnimelech et al., 1994; Crab et al., 2007; Avnimelech, seawater was added to 150 ml of SSAC. The samples were incubated at
2009). 30 °C in a 150-rpm shaker. The concentrations of ammonia nitrogen and
With the development and improvement of BFC technology, het- nitrite were measured by the μMAC SMART hydraulics rev.3 instrument
erotrophic nitrifying and ammonia assimilation bacteria have garnered at 0, 3, 6, 9, 12, 24, 36 and 48 h. The experimental and control group
increasing attention in the field of aquaculture (Ebeling et al., 2006; had three replicates each.
Wasielesky et al., 2006; Avnimelech, 2009). In this study, a hetero-
trophic Sphingomonas sp. strain that can remove both ammonia nitrogen 2.5. The effect of the selected strain on nitrite removal
and nitrite was isolated and their potential values for the treatment of
farming waste water were assessed. The effect of the selected strain on ammonia nitrogen removal was
determined with the same method described in section 2.4 except the
nitrogen source was substituted with NaNO2 and the concentration of
2. Materials and methods
nitrite in the water samples was adjusted to 5 mg/l.
2.1. Medium preparation and sample collection
2.6. Bio-safety assessment of the bacterial strain to shrimp Litopenaeus
vannamei
Isolation medium for ammonia removal bacteria (IMAR) containing
1 g of (NH4)2SO4, 5 g of glucose, 5 g of sucrose, 0.2 g of yeast extract,
Litopenaeus vannamei was chosen to assess the biosafety of the bac-
and 0.5 g of NaH2PO4, in 1 l of filtered seawater was prepared, adjusted
terial strain since it is one of the most economically important aquatic
pH to 7.0 and sterilized. Concentrated IMAR (10×, CIMAR) was also
species. Healthy shrimp (4 ± 1 cm) from an aquatic farm in Shunde
prepared for enrichment of the ammonia removal bacteria. Nitrite-
(Guangdong Province, China) were temporarily cultured for five days in
utilizing medium (NUM) containing 1 g of NaNO2, 5 g of glucose, 5 g of
artificial seawater (10‰) prepared with sea salt and then the shrimp
sucrose, 0.2 g of yeast extract, and 0.5 g of NaH2PO4, in 1 l of filtered
were randomly grouped into tanks with 5-l artificial seawater disinfected
seawater was prepared, adjusted pH to 7.0 and sterilized. The water
by chlorine dioxide (South Ranch, China). Every group contained eight
samples were collected from the shrimp culture ponds in Maoming,
shrimps. The selected strain was cultured overnight and the optical
Guangdong Province, China, and stored at 4 °C before use.
density of the cells was adjusted to OD600nm = 1.0 (approximately
5 × 108 cfu/ml). Then 50 ml of bacterial fluid was centrifuged, re-
2.2. Isolation and screening of bacteria suspended in 5 ml of sterilized seawater, and washed twice with the same
volume of sterilized seawater. In the experimental group, 0.5 ml of sus-
The water samples were added into CIMAR at a ratio of 9: 1 in 250- pended bacterial cells was added into each tank, whereas in the control
ml conical flasks and then incubated at 30 °C in a shaker to enrich the group, 0.5 ml of sterilized seawater was added into each tank. Both the
bacteria that can utilize ammonia nitrogen and organic carbon sources. experimental group and the control group had three replicates each.
After 48 h of enrichment, 100 μl of bacteria was spread on Luria-Bertani During the period of the experiment, the shrimp were fed normally, and
(LB) agar plates and incubated overnight at 30 °C. Single colonies were seawater was completely changed each day. After the water change,
picked based on their colony morphologies and inoculated in culture 0.5 ml of bacterial cells newly prepared with the abovementioned
tubes with 4 ml of IMAR for 12 h, and then cultured bacteria were method was added into each tank in the experimental group whereas in
streaked on IMAR agar plates three times for purification. A total of 20 the control group it was substituted by 0.5 ml of sterilized seawater. The
bacterial strains were obtained in this way. Then, 100 μl of each strain number of survival shrimps was recorded for seven days.
that was cultured for 12 h was added into 4 ml of IMAR and incubated
on in a 200-rpm shaker for 48 h. The initial and the final concentration 2.7. The impact of the strain on the growth of Vibrio spp.
of ammonia‑nitrogen in the different culture mediums were determined
by a μMAC SMART hydraulics rev.3 instrument. The analysis included 2.7.1. Inhibition test on the number of Vibrio spp. in farming water
three replicates. The strain with the most removed ammonia nitrogen An experiment was conducted to learn whether the strain can in-
was preserved and used for subsequent experiments. hibit the growth of Vibrio species in cultured water. A water sample was

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taken from the culture pond experienced an outbreak of vibriosis and Sphingomonas. The 16S rDNA sequence of Sphingomonas sp. LPN080 was
the number of Vibrio cells was calculated by conventional TCBS plates deposited in GenBank under the accession number, MH392718.
(Oliver et al., 1983). A total of 2 l of the water sample was added with
2 g of glucose. Then, 150 ml of the water sample containing glucose was 3.2. Ammonia nitrogen removal by Sphingomonas sp. LPN080
added into each 250-ml conical flask. Each conical flask in the experi-
mental group was added with 100 μl of bacterial fluid (OD600nm = 1.0) After 48 h of shaker culture, the concentration of the ammonia ni-
whilst each conical flask in the control group was added with 100 μl of trogen of water samples was reduced from an initial 8 mg/l to 0.3 mg/l,
sterilized seawater. The experimental group and control group each and the degradation rate of ammonia nitrogen was up to 96%.
contained four replicates. After 48 h of incubation at 30 °C in a 150-rpm However, in the control group, the concentration of ammonia nitrogen
shaker, the water samples were serially diluted (105 dilution), spread of water samples nearly remained unchanged (Fig. 3). In addition, the
on fresh TCBS plates, and incubated at 30 °C overnight. The colonies on concentration of nitrite was also tested during the whole monitoring
the plates (mainly representing Vibrio colonies) were numbered and process, and the result showed that the concentration of nitrite could
recorded (Kobayashi et al., 1963). not be detected.

2.7.2. Plate suppression experiment of the strain 3.3. Nitrite removal by Sphingomonas sp. LPN080
To directly exhibit whether the strain can inhibit the growth of
Vibrio species, we conducted plate suppression experiment. Four Vibrio The concentration of nitrite in the experimental group was reduced
strains, V. alginolyticus E06333, V. furnissi ATCC 33813, V. harveyi E385, from an initial 5 mg/l to 0.8 mg/l and the removal rate was approxi-
and V. parahaemolyticus ATCC 17802, were used. 100 μl of Vibrio cells mately 81% in 48 h, whereas the concentration of nitrite in the control
were spread onto LB plates and then each plate was covered with three group remained nearly unchanged (Fig. 4). This indicated that the
5-mm sterilized filter sheets. The filter sheets were dropped with 5 μl of LPN080 strain also had a strong ability to remove nitrite in water
tested cells (Barry et al., 1979). The sizes of the inhibition zones around samples. However, the removal rate of nitrite was lower than that of
the filter sheets were observed and measured. ammonia nitrogen by the LPN080 strain (96%). The concentration of
ammonia nitrogen was also tested during the monitoring process, and
3. Results the result indicated that only low concentration of ammonia nitrogen
(< 0.1 mg/l) could be detected.
3.1. Isolation and identification of potential probiotic strains
3.4. Assessment of biological safety for L. vannamei
A total of 20 bacterial strains that can grow in IMAR containing
ammonia nitrogen and LB plates were preliminarily isolated and pur- The survival rate of shrimps cultured with or without the LPN080
ified. By comparing of ammonia removal efficiency, all of the 20 strains strain is shown in Fig. 5. The accumulative survival rate of shrimp
obtained had different levels of ammonia removal activities (Fig. 1). (83.3%) in the experimental group was only slightly higher than that
After 48-h incubation, the lowest concentration of ammonia nitrogen (79.2%) in the control group, and thus, there is no significant difference
was observed in IMAR medium containing the cells of the LPN080 between the experimental and control groups. Considering a big dosage
strain (Fig. 1). A total of 47.6% of ammonia nitrogen in the IMAR was of LPN080 in the experimental group (approximately 5 × 105 cfu/ml),
removed by the LPN080 strain over 48 h (from 210.5 mg/l to 110.2 mg/ the result indicates the LPN080 strain has high biosafety for L. vannamei.
l). As a result, the LPN080 strain was screened out as a candidate strain
for the subsequent experiments. 3.5. Inhibition of the LPN080 strain to the growth of Vibrio spp.
The BLASTN search indicated that the16S rDNA sequence of the
LPN080 strain had 99% similarity with many strains from the genera The inhibitory activities of the LPN080 strain to the growth of Vibrio
Sphingomonas. A sequence comparison in the Ribosomal Database Project species was tested by two methods. The TCBS plate count indicated that
(RDP) database gave the same result. The phylogenetic tree based on 16S the number of Vibrio cells in the primary farming water was approxi-
rDNA sequences of the LPN080 strain and the sequences of related mately 1.1 × 105 cfu/ml. When LPN080 cells were added into farming
bacterial strains clearly revealed that all of the Sphingomonas strains in- water samples supplemented with glucose (which served as the ex-
cluding LPN080 clustered into one large clade (Fig. 2). These results perimental group), the growth of Vibrio spp. was strongly inhibited as
roughly manifested that the LPN080 strain is one member of the genera almost no bacterial colonies formed on the TCBS plates (105 dilution)

Fig. 1. A comparison of ammonia nitrogen removal by different bacterial strains. Control: IMAR medium without the addition of bacterial cells.

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Fig. 2. A constructed phylogenetic tree based on 16S rDNA sequences of the LPN080 strain and related bacterial strains.

Fig. 3. The effect of the LPN080 strain on ammonia nitrogen removal. EG: water samples in the experimental group inoculated with the LPN080 strain; CG: water
samples in the control group without the addition of the LPN080 strain.

Fig. 4. The effect of the LPN080 strain on nitrite removal. EG: water samples in the experimental group inoculated with the LPN080 strain; CG: water samples in the
control group without the addition of the LPN080 strain.

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and nitrite are mainly removed by various beneficial bacteria and patho-
gens are also inhibited by these consciously cultured bacteria
(Avnimelech, 2005; Avnimelech, 2009; Balcázar et al., 2006). The success
of BFC manifests that probiotic bacteria have great development potential
to control water quality, to inhibit pathogens, and to reduce the water
exchange. Therefore, isolating the potential probiotic bacteria with the
aim of applying them in BFC is an urgent and interesting goal.
In the models of traditional BFC, ammonia nitrogen and nitrite are
generally removed by the nitrification and denitrification reactions of
bacteria (Brune et al., 2004), and little attention has been focused onto
the bacteria possessing the ability of ammonia assimilation. Since am-
monia assimilation by bacteria can directly absorb ammonia nitrogen
and convert it into the cellular components of bacteria (Avnimelech,
1999; Wang and Tan, 2013), theoretically it has a higher efficiency to
Fig. 5. Survival of shrimps after immersion challenge by the LPN080 strain. EG: remove ammonia nitrogen compared to conventional nitrification and
the experimental group challenged by the LPN080 strain; CG: the control group denitrification reactions of bacteria comprised of several biochemical
without the challenge by the LPN080 strain. steps. In the present study, Sphingomonas sp. LPN080 can efficiently re-
move ammonia nitrogen over a short time without the accumulation of
nitrite. The inexistence of nitrite suggests that it is impossible that am-
whereas the number of Vibrio spp. remained at a normal level (aver-
monia nitrogen is transformed into nitrite by the usual nitrification
agely 13.5 colonies per plate) in the control group (Fig. 6). To further
process. Since no nitrogen sources were offered in the medium except
explore what caused the inhibition to the growth of Vibrio spp., a plate
ammonia nitrogen, there is only one possibility; Sphingomonas sp.
suppression experiment of the LPN080 strain was also conducted, and
LPN080 can assimilate ammonia nitrogen into its cellular components.
the results showed that the growth of LPN080 cells on filter sheets could
The activity of ammonia assimilation has been recorded in many bac-
not inhibit the expansion of four Vibrio cells as no inhibition zones
teria, such as Azorhizobium caulinodans (Michel-Reydellet and Kaminski,
occurred (Fig. 7). These results clearly indicated that the inhibition to
1999), Corynebacterium glutamicum (Burkovski, 2003), and rumen bac-
Vibrio spp. was not likely caused by the antimicrobial substances that
teria (Wang and Tan, 2013), but had not been previously observed in
were supposedly generated by the LPN080 cells.
Sphingomonas spp. Therefore, this is the first report that a strain of
Sphingomonas may have the ability to assimilate ammonia nitrogen.
4. Discussion Notably, Sphingomonas sp. LPN080 also can efficiently remove nitrite
and only a very low concentration of ammonia nitrogen was detected
The accumulation of ammonia nitrogen and nitrite are two major toxic during this process. Likewise, in this case, nitrite was the only nitrogen
factors that directly affect the economic benefits of aquaculture (Alcaraz source for the growth of Sphingomonas sp. LPN080. Since nitrite cannot be
et al., 1999; Körner et al., 2001; Ebeling et al., 2006), and thus how to converted into ammonia by bacteria, we assume that the nitrite was first
control the concentrations of ammonia nitrogen and nitrite in aquaculture nitrified into nitrate and that the nitrate was then likely absorbed by the
water represents an important research interest. Plants and algae (mac- LPN080 strain. The genus Sphingomonas was defined in 1990 (Yabuuchi
rophytic algae or microalgae) show high efficiency for the removal in- et al., 1990) and the bacteria in the genus are frequently reported to have
organic nitrogen (Egashira and Sato, 2007; Habaki et al., 2011; Sánchez- prominent degradation capabilities for various environmental con-
Romero et al., 2013), however they either occupy the living space of taminants (e.g. Kirimura et al., 1999; Coughlin et al., 1999; Perruchon
farming aquatic animals or release harmful substances after their un- et al., 2016). However, there are no current reports that focus on whether
controllable growth and death (Liu et al., 2009). Farming using RAS can bacteria in Sphingomonas have the activities of ammonia and nitrite ni-
tightly control water quality (Martins et al., 2010; Timmons et al., 2002), trogen removal. Tal et al. (2003) characterized a nitrifying microbial
but the large cost of RAS limits its expansion within the industry, espe- consortium from a moving bed bioreactor connected to a marine RAS
cially in developing countries. In recent years, biofloc farming has become using denaturing gradient gel electrophoresis of amplified 16S rRNA gene
an environmentally friendly farming model, in which ammonia nitrogen fragments. Sphingomonas spp. were detected together with other ammonia-
and nitrite-oxidizing bacteria, which suggests the capability for nitrifica-
tion, denitrification and anammox in the system. Certain strains of
Sphingomonas were also found to contribute to ammonia oxidation under
low dissolved oxygen in a wastewater treatment plant (Fitzgerald et al.,
2015). Based on our results and a few reports, we speculate that at least
some members in Sphingomonas could transform the different forms of
inorganic nitrogen, which may serve as the only nitrogen source. Nu-
merable bacteria can utilize nitrate as the sole nitrogen source (Lin and
Stewart, 1998; Moreno-Vivián and Flores, 2007), and along with the de-
velopment of molecular and sequencing techniques, bacteria capable of
assimilating nitrate have been demonstrated to play an important role in
the nitrogen cycle (Moreno-Vivián and Flores, 2007; Luquealmagro et al.,
2011). A complete genome sequencing of Sphingomonas sp. LPN080 is
ongoing, and it will increase our understanding of nitrogen metabolism in
the strain and explain the phenomenon observed in this study.
The existence of Sphingomonas species in farming environments and
aquatic animals has not received widespread attention, and only a few
Fig. 6. Number of colonies on the TCBS plates with the same dilution. EG: species (or strains) from Sphingomonas have been reported to be isolated
water samples supplemented with the LPN080 cells and glucose; CG: water from or exist in farming environments and aquatic animals (Tal et al.,
samples supplemented with glucose. Each sample in experimental and control 2003; Floris et al., 2013; Alipiah et al., 2016; Chaudhary and Qazi, 2014;
groups has three independent dilutions, spreading, and numbering (n = 12). Chen et al., 2016; Wang et al., 2018). A Sphingomonas strain, AsCh-P3,

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Fig. 7. Inhibition test of Sphingomonas sp. LPN080 against four Vibrio species. A: V. alginolyticus E06333; B: V. furnissi ATCC 33813; C: V. harveyi E385; D: V. parahaemolyticus
ATCC 17802; −: negative control (added with the medium); +: positive control (added with chloramphenicol); and the white circles in the picture are filter sheets.

showed strong antagonistic activity against the fish pathogen V. angu- to Sphingomonas sp. LPN080 and this is not the only case. We observed in
illarum during in vitro and in vivo assays (Chaudhary and Qazi, 2014), BFC that the number of Vibrio bacteria gradually decrease to a very low
which suggests the existence of antibacterial substances generated by level (< 102 cfu/ml) when glucose or sucrose is used as carbon supple-
AsCh-P3. In our study, Sphingomonas sp. LPN080 can inhibit the growth ment (unpublished data). Little is known about the relationship between
of Vibrio species in seawater when supplemented with glucose but it heterotrophic bacteria and Vibrio bacteria in the water environment
cannot produce growth-inhibitory substances against four tested Vibrio when carbon sources are manually added, and thus many mechanisms of
cells grown on LB plates. The phenomenon may be explained by the microbial antagonism need to be discovered in future.
speculation that the addition of glucose changed the carbon/nitrogen
ratio and glucose was preferentially utilized by Sphingomonas sp. LPN080 5. Conclusions
rather than Vibrio spp. In addition, the decreased pH in water (data not
shown) may be more disadvantageous to the growth of Vibrio spp. than Sphingomonas sp. LPN080 can not only efficiently assimilate

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ammonia nitrogen and remove nitrite in farming water but also in- sterile Ulva sp. for water quality control of intensive shrimp culture ponds in devel-
directly inhibit the growth of Vibrio spp. in water when supplemented oping countries. Chem. Biochem. Eng. Q. 25, 341–349.
Haseeb, M., 2012. Development of Zero Water Exchange Shrimp Culture System
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