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FENG Yuwei, SU Xinguo, SUN Huiming, LIN Haopeng, CHEN Qionghua, SHU Hu. Identification and denitrification performance of a high ammonia nitrogen-resistant aerobic denitrifying bacteria[J]. South China Fisheries Science, 2023, 19(6): 107-115. DOI: 10.12131/20230079
Citation: FENG Yuwei, SU Xinguo, SUN Huiming, LIN Haopeng, CHEN Qionghua, SHU Hu. Identification and denitrification performance of a high ammonia nitrogen-resistant aerobic denitrifying bacteria[J]. South China Fisheries Science, 2023, 19(6): 107-115. DOI: 10.12131/20230079

Identification and denitrification performance of a high ammonia nitrogen-resistant aerobic denitrifying bacteria

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  • Received Date: April 15, 2023
  • Revised Date: September 03, 2023
  • Accepted Date: September 21, 2023
  • Available Online: June 20, 2023
  • Ammonia (NH4 +-N), nitrate (NO3 -N) and nitrite (NO2 -N) are the main contaminants in industrial aquaculture systems. High nitrogen concentration in water is likely to cause aquaculture water pollution and endanger the safety of aquatic animals. Aerobic denitrifying bacteria are widely used to remove nitrogen-containing aquaculture wastewater. In order to obtain a strain that can safely and efficiently treat wastewater with high ammonia nitrogen concentration, we studied the aerobic denitrifying bacteria WM28 with high ammonia nitrogen resistance screened from aquaculture ponds. The strain was identified through morphological observation, physiological and biochemical tests and 16S rRNA gene sequencing. The environmental and biological safety of the strains were evaluated through antibiotic tests and zebrafish (Danio rerio) toxicity tests. The growth and denitrification performance were measured in three single nitrogen source simulated wastewater, and the denitrification capacity was tested in high concentration ammonia nitrogen simulated wastewater. WM28 was identified as Rhodococcus ruber with high antibiotic sensitivity and good biosafety. The removal rates were 100%, 76.3% and 66.99% after 48 h incubation in single NH4 +-N, NO3 -N and NO2 -N media, respectively. Their removal rates of NH4 +-N reached 100% at high concentrations of 100–500 mg·L−1 NH4 +-N in simulated wastewater experiment after 48 h. 700 mg·L−1 NH4 +-N was removed by more than 88% after 116 h. At 120th hour, the initial NH4 +-N concentration of 1 000 mg·L−1 was still capable of denitrification with a removal rate of 74.38%, which indicates that strain WM28 has great tolerance to high ammonia nitrogen. In summary, strain WM28 is a safe and efficient aerobic denitrifying bacteria with high ammonia tolerance, and has promising application prospects in the treatment of aquaculture and industrial wastewater.

  • Larimichthys crocea is a class of vital commercial fishes in China, with the mariculture production being up to 257 683 t, ranking first among the mariculture fishes for consecutive years [1]. However, with the rapid increase in production, environmental degradation, and the intensification of farming practices, issues related to quality deterioration—such as flavor deterioration and declining meat quality—have emerged, thereby reducing market acceptance [2-4]. Ma et al. [5] found that the farmed L. crocea significantly differed from the wild in the body shape, color, muscle texture and flavor. Therefore, in the mariculture of L. crocea, enhancing muscle quality while maintaining production levels has become a key concern for the industry.

    Muscle quality is a complex concept and is evaluated generally based on its flavor, taste, nutritive value and safety ,[6] while delicate flavor, an important factor in determining the muscle flavor, primarily depends on the two substances of flavor amino acids (glutamic acid and amino acid amide mainly) and nucleotides (inosine monophosphate, IMP, and guanosine monophosphate, GMP) [7]. IMP has an umami level nearly 40 times the value of glutamic acid and shows a synergistic effect with the latter to increase its level of umami by 30 folds [8]. Compared with IMP, GMP has a very low content with a limited contribution to the umami. As a result, IMP is considered a key umami compound in aquatic animals and serves as an important indicator for evaluating muscle quality [9-10]. Various factors, including species, sex, climatic conditions, farming environment, feeding practices, age and body mass of farmed animals, as well as post-slaughter storage conditions, can all influence the IMP content in muscle products [11-12].

    IMP, also known as hypoxanthine nucleotide, has two routes of synthesis in living organisms, that is, "denovo synthesis" as the primary route in the body, and "salvage synthesis" which mainly occurs in marrow, brain and other tissues [7]. The former involves more than ten enzymes, in which, adenylosuccinate lyase (ADSL) is a bifunctional enzyme catalyzing the initiation of purine nucleotide synthesis and its cycle, which catalyzes the eighth step of "denovo synthesis" and makes adenylosuccinic acid (monophosphate) be transformed into adenylic acid (monophosphate) to exert a key regulatory effect in the synthesis process of IMP [13-14]. At present, poultry and livestock are mainly used to investigate the characteristics of adsl gene and the correlation between it and the content of IMP in farmed animals. Xu et al. [15] found that there was a significant positive correlation between the expression level of adsl gene and the corresponding IMP content in different parts of ducks regardless of their varieties and sex. Chen et al. [16] found an extremely significant positive correlation between the content of IMP and the expression of adsl gene among different pig breeds. The adsl gene of Danio rerio [17] and Ctenopharyngodon idella [18] in aquatic animals had been cloned, but relevant research focuses on its molecular characteristics and there is no report on the correlation between its expression level and the content of IMP. In the present study, we cloned the sequence of adsl gene in L. crocea, analyzed the genetic structure and evolutionary characteristics and determined its expression levels in different tissues by adopting real-time quantitative PCR (qPCR). Meanwhile, the content of IMP and the expression level of adsl gene in the muscle tissues of L. crocea cultured in different sizes were tested. The research results may lay the foundation for subsequent in-depth investigations on the mechanism of action of adsl gene in IMP synthesis and flavor formation in L. crocea.

    The healthy L. crocea with different farming sizes were obtained from the same offshore cage located in Sanduao, Ningde, Fujian Province, to ensure consistent rearing conditions and feeding practices. Three groups were selected based on average body mass: Group 1 [(243.08 ± 7.32) g], Group 2 [(281.80 ± 6.70) g], and Group 3 [(460.44 ± 19.86) g], with five fish per group. All fish were anesthetized using MS-222 and dissected on ice. For Group 1, samples of muscle, heart, liver, gill, brain, anterior intestine, mid-intestine, posterior intestine, stomach, skin, kidney, spleen, and head kidney were collected for tissue-specific gene expression analysis. For Groups 2 and 3, only muscle tissue was sampled. Muscle samples from all three groups were divided into two portions: one for total RNA extraction and the other for IMP content determination. All collected tissues were immediately frozen in liquid nitrogen and then stored at –80℃ until further analysis.

    Total RNA was extracted from the muscle tissue of L. crocea using the Eastep® Super Total RNA Extraction Kit (Promega, USA), following the manufacturer's instructions. The quality of the extracted RNA was assessed by 2% (w) agarose gel electrophoresis, and its concentration was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized using the Eastep® RT Master Reverse Transcription Kit (Promega, USA) according to the manufacturer's protocol. The resulting cDNA was stored at –20℃ for subsequent use.

    Based on the predicted adsl gene sequence of L. crocea available in GenBank (accession No. XM_019265987.2), a pair of primers, Lcadsl-F/R (Table 1), was designed using Primer 5 software to amplify the open reading frame (ORF) of the gene. PCR amplification was performed using cDNA from muscle tissue as the template, with an annealing temperature of 55℃. The amplified product was verified by 2% (w) agarose gel electrophoresis, and the target band was recovered using a gel extraction kit. The purified PCR fragment was then ligated into the pMD-19T vector (TaKaRa, Japan) and transformed into Escherichia coli DH5α competent cells. Positive clones were screened by ampicillin resistance and colony PCR, and confirmed by sequencing conducted at Fuzhou Boshang (Shangchen) Biotechnology Co., Ltd.

    Table  1  Primers used for gene cloning and qPCR of adsl in L. crocea
     | Show Table
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    NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/), an online tool, was used to analyze the ORF of adsl gene in L. crocea and deduce its amino acid sequence. Expasy (https://web.expasy.org/compute_pi/) was adopted for analysis of the molecular weight of its protein and the isoelectric point, and SMART software ((http://smart.embl-heidelberg.de/) for prediction of the structural domain of its protein. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), an online comparison tool of NCBI, was employed for homology analysis of ADSL in L. crocea. ADSL sequences from different species were selected for multiple sequence alignment, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0. Splign (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi) was used to analyze the genetic structure of adsl in L. crocea.

    To explore the tissue-specific expression pattern of adsl mRNA in L. crocea, total RNAs were extracted from various tissues of five fish from Group 1 following the method described in Section 1.2, and subsequently reverse-transcribed into cDNA template. Primers specific to the adsl gene were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 1). qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (2×) (Vazyme, China) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA). The housekeeping gene β-actin was used as an internal reference [19] (Table 1), and each sample was analyzed in triplicate. The qPCR program was as follows: 95 ℃ for 30 s; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 30 s, 40 cycles in total. A melting curve analysis was performed at the end of the amplification to confirm primer specificity. The relative expression level of adsl mRNA in different tissues was calculated using the 2−ΔΔCt method based on the Ct values of the target gene adsl and internal gene β-actin[20].

    According to the methods described by Jiang et al. [21] and Liu et al. [22], the content of IMP in the muscle tissues of L. crocea was determined using high-performance liquid chromatography (HPLC). IMP standard solutions (Shanghai Yuanye Bio-Technology Co., Ltd.) were prepared by gradient dilution with a mobile phase consisting of phosphate buffer and methanol at a volume ratio of 1 000:40 [V(phosphate buffer): V(methanol)], to obtain concentrations of 1.00, 5.00, 10.00, 50.00, 100.00, and 200.00 mg·L−1. The resultant solutions were filtered through a 0.22 μm microfiltration membrane and sonicated for 30 min. A 10 μL aliquot of each sample was injected into an HPLC system (Agilent, USA) equipped with an Agilent C18 column (4.6 mm × 250 mm, 5 μm) and a diode array detector (DAD). The mobile phase flow rate was set at 1.0 mL·min−1, and the detection wavelength was 254 nm. A standard calibration curve was established based on the peak areas corresponding to different IMP concentrations, which was then used to quantify the IMP content in muscle tissue samples of L. crocea.

    Muscle tissue from L. crocea of different farming sizes was accurately weighed and homogenized. To the homogenized tissue, 5 mL of 10% (φ) perchloric acid was added, and the mixture was subjected to ultrasonic treatment (Sonics, USA) for 30 minutes. The resulting solution was then centrifuged to collect the supernatant. An additional 5 mL of 5% (φ) perchloric acid was added to the precipitate, and the supernatant was obtained using the same procedure. The two supernatants were combined, and the pH of the solution was adjusted to 6.5 using potassium hydroxide (KOH). The solution was then diluted to a final volume of 20 mL with ultrapure water and filtered through a 0.22 μm microporous membrane before being analyzed. The detection parameters were consistent with those used in the standard curve establishment. The IMP content in the samples was calculated based on the peak area obtained for each sample, using the standard curve equation. The calculation formula is as follows:

    W=[(CC0)×V×N]/m

    where W is the mass fraction of IMP in the muscle (mg·kg−1); C is the mass concentration of IMP in the test solution (mg·L−1); C0 is the mass concentration of IMP in the blank control (mg·L−1), which is 0; V is the stated volume, i.e., 20 mL; N is the dilution ratio, which is 1; m denotes the muscle mass weighed in each group (g).

    Total RNAs were extracted from the muscle tissues of L. crocea in groups 1, 2 and 3 according to the method described in Section 1.2, and reverse-transcribed into cDNA template. qPCR was used to determine the expression of adsl mRNA, with the detailed method in Section 1.4. The 2−ΔΔCt method was adopted to calculate the relative expression level of adsl in muscle tissues of L. crocea with different sizes.

    The experimental data were expressed as mean ± standard error (ˉx±sˉx). SPSS Statistics 27 was adopted for one-way ANOVA and Duncan's multiple comparisons on the tissue distribution of adsl mRNA in healthy L. crocea and the expression differences of adsl mRNA among the three groups and the corresponding IMP content, with a significance level of α = 0.05. Pearson correlation analysis was conducted to examine the relationship between adsl gene expression and IMP content, and P < 0.05 indicated a linear correlation on the whole and Pearson correlation coefficient r > 0 meant a positive correlation between them. GraphPad Prism 8 was used for drawing.

    Using cDNA of the muscle tissues in L. crocea as the template, the ORF of the adsl gene was amplified by PCR, yielding a full-length sequence of 1 446 bp that encodes a polypeptide of 481 amino acids (Fig. 1). The sequence had been submitted to NCBI database with the accession number of OQ126879.1. The resulting protein (ADSL) had a relative molecular weight of 54.6 kD and an isoelectric point of 6.19, including two primary structure domains: N-terminal lyase 1 (Pfam Lyase_1) (Ala24–Leu309) and C-terminal ADSL_C (Leu374–Leu458) domains (Fig. 2). The lyase_1 domain also contains a typical fumarase family motif (Ser286–Glu299) (Fig. 1).

    Fig. 1  Nucleotide and amino acid sequence of adsl gene in L. crocea
    Note: The Pfam Lyase_1 domain is shown with red underlines. The ADSL_C domain is shown with blue underlines. The fumarase region is shaded in grey.
    Fig. 2  Prediction of ADSL conserved structure domain in L. crocea

    A homology comparison was carried out between the amino acid sequence of ADSL in L. crocea and that of the other species. The results showed a high similarity to teleosts such as Collichthys lucidus (TKS91682), Sparus aurata (XP_030263577.1), Oryzias melastigma (KAF6719572.1), C. idella (ACB72735.1) and Danio rerio (NP_956193.2), with the values being up to 98.54%, 95.63%, 93.14%, 92.53% and 91.08%, respectively; the similarity to Xenopus tropicalis (NP_001005457.1) and Gallus gallus (NP_990860.2) was 76.74% and 78.97%, respectively; the similarities to mammals, including Mus musculus (NP_033764.2), Sus scrofa (ADA82235.1), Bos taurus (NP_001095847.1) and Homo sapiens (AAC21560.1), were relatively low, which were 76.24%, 77.07%, 75.10% and 75.83%, respectively.

    According to the phylogenetic tree, L. crocea clustered with C. lucidus into a distinct clade with a high bootstrap value (98%), further grouping with other marine teleosts into a single branch, and then with freshwater species such as Cyprinus carpio into a large clade (Fig.3), consistent with the results of homology analysis. From the genetic structure, adsl gene of L. crocea showed a structural organization similar to that of marine species such as Acanthopagrus latus and Dicentrarchus labrax, all comprising 12 exons and 11 introns. In contrast, freshwater fishes like C. idella and other higher organisms possess adsl genes consisting of 13 exons and 12 introns (Fig. 4), suggesting the structural changes of adsl gene in the evolution process.

    Fig. 3  Phylogenetic analysis of different species based on ADSL amino acid sequences
    Fig. 4  Structure analysis of adsl genomic DNA sequence in different species

    qPCR was adopted to determine the tissue distribution of adsl mRNA in healthy L. crocea. The amplification efficiencies of adsl and β-actin genes were calculated through gradient dilution of the template and establishment of the standard curve, yielding efficiency values between 0.95 and 1.05. The presence of a single peak in the melting curve confirmed the specificity of the amplification. Among the examined tissues, adsl mRNA expression was highest in muscle, significantly exceeding that in all other tissues (P < 0.05), followed by the heart, while the remaining tissues exhibited low expression levels (Fig. 5).

    Fig. 5  qPCR analysis of tissue distribution of adsl transcripts in healthy L. crocea
    Note: Different letters represent significant differences between different tissues (P < 0.05).

    The IMP content in the muscle tissue of cultured L. crocea with different sizes was determined using HPLC. A linear equation was established by gradient dilution of the IMP standard, yielding the regression formula y = 0.2715x − 0.0734 with a determination coefficient (R2) of 0.999 8, indicating a strong fit suitable for subsequent experimental analysis (Fig. 6-a). The standard curve established was used to determine the mass fraction of IMP in the muscle of cultured L. crocea with different sizes (Fig. 6-b). Group 1 showed an IMP content of (2 663.93±54.52) mg·kg−1; Group 2 had a significantly higher content of (2 996.43±24.10) mg·kg−1 (P < 0.001); Group 3 reached (3 313.00±54.89) mg·kg−1, which was significantly higher than those in groups 1 and 2 (P < 0.01).

    Fig. 6  IMP content in muscles of L. crocea with different sizes
    Note: a. Construction of a standard curve to determine the IMP content; b. IMP content in muscles of L. crocea with different sizes; **. P < 0.01; ***. P < 0.001.

    qPCR was adopted to determine the relative expression level of adsl gene in the muscle of cultured L. crocea with different sizes. The results showed that compared with Group 1 (L. crocea with small size), Group 2 had a significantly increased expression level of adsl mRNA in the muscles (P < 0.01), 3.11 times the value of Group 1, and the value in Group 3 was significantly higher than those in groups 1 and 2, which was 4.95 times the value of Group 1 (Fig. 7-a).

    Fig. 7  Relative expression of adsl m RNA in muscles of L. crocea with different sizes (a) and its correlation analysis with IMP content (b)
    Note: ***. P < 0.001.

    As shown in Fig. 7-b, the variation of adsl gene expression in the muscles of L. crocea with different sizes was consistent with the trend observed in IMP content. Pearson correlation analysis revealed a statistically significant linear relationship between adsl expression and IMP content (P < 0.000 1), with a correlation coefficient of r = 0.962, indicating a strong positive correlation between the two variables.

    As ADSL plays a crucial part in purine nucleotide biosynthesis and cycling [23], it is foundational for the investigation of the synthesis mechanism of IMP to analyze the molecular and expression characteristics of ADSL. ORF of adsl gene in L. crocea cloned in this study had a full-length sequence of 1 446 bp that encodes a polypeptide of 481 amino acids. The molecular size of this gene is comparable to that of C. idella[24]. The ADSL protein contains two conserved domains: N-terminal lyase 1 and C- terminal ADSL_C, which is consistent with the structure of ADSL of other species such as goose [14] and donkey [23]. Studies have demonstrated that Class Ⅱ fumarase, aspartase, argininosuccinate lyase and ADSL superfamily all contain a conserved domain consisting of 14 amino acids "SSxxPxKxNxxxxE" [18, 25]. while the typical fumarate domain of L. crocea is "SSAMPYKRNPMRAE". Homology analysis revealed that ADSL in L. crocea shares over 90% amino acid sequence identity with other teleosts, while the similarity drops to just above 75% when compared with amphibians, birds, and mammals. The antibody against human ADSL protein could specifically recognize ADSL of C. idella (Yuan et al. [18]). All these demonstrated a high conservation of ADSL. In terms of genetic structure, the adsl gene in higher organisms including M. musculus and H. sapiens consists of 13 exons and 12 introns, which was the same as that of freshwater fishes including C. idella and D. rerio. In contrast, L. crocea and other marine fishes possess an adsl gene composed of 12 exons and 11 introns, indicating that structural changes occurred in the evolution process.

    ADSL is a key enzyme in purine nucleotide metabolism and plays a critical role in the energy metabolism of eukaryotic organisms. As a result, it exhibits high activity in tissues such as muscle and brain [26]. In C. idella, the muscle showed the highest expression level of the adsl gene, followed by the heart and brain tissues, indicating that these tissues are involved in the most active purine nucleotide synthesis [18]. In L. crocea, adsl mNA showed a constitutive expression in different tissues, with the highest expression observed in the muscle, followed by the heart. Expression levels in other tissues are relatively lower, which is generally consistent with the pattern observed in C. idella. However, in C. idella, the relative expression of this gene showed small differences across different tissues, with the value in the muscle being about 2.5 times that in the heart and 3 to 7 times the value in other tissues. In contrast, in L. crocea, the muscle expression level was 15.8 times the value in the heart and nearly 100 times that in other tissues like the brain. These differences might be associated with factors such as fish species, fish size and farming environment, with the specific reason requiring further research.

    ADSL plays a crucial role in the synthesis of the muscle flavor compound IMP. Many studies have shown a positive correlation between the expression of adsl gene in the muscles of farmed animals and the IMP content in muscles [15-16, 27]. In this study, the HPLC method was adopted to determine the IMP content in the muscle of L. crocea with different sizes under the same farming conditions. The results indicated that the IMP content in the muscle tissue was significantly higher in fishes with larger farming sizes than in those with smaller sizes (P < 0.01), and the expression of the adsl gene followed a similar trend. Correlation analysis revealed a significant positive correlation between the two. These findings suggest that, similar to higher animals like livestock and poultry, the expression of the adsl gene in the muscle tissue of L. crocea is positively correlated with the IMP content. In the present study, we only sampled three size groups of farmed L. crocea, and future research will focus on analyzing the expression of the adsl gene and the corresponding IMP content at different growth stages within the same batch of L. crocea. Furthermore, aside from fish size, farming environment and feed composition also significantly influence IMP synthesis and the expression of related metabolic genes, which in turn affect the IMP content in the muscle [11, 28]. Therefore, to ensure the accuracy of the results, all fish sampled in this study were sourced from the same farming net cage.

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