1 s2.0 S0961953420302117 Main
1 s2.0 S0961953420302117 Main
1 s2.0 S0961953420302117 Main
A R T I C L E I N F O A B S T R A C T
Keywords: Bioenergy from biomass crops plays an important role as a promising energy source to meet the energy demands.
Bacillus strains However, during the production of biomass crops, water requirement, water quality degradation and the
Nitrogen removal resulting environmental and economic problems are important factors that can affect the development of bio
Biological treatment
energy worldwide, especially in low- and middle-income countries. In addition, industrial-scale production of
Aerobic denitrification
Wastewater
bioenergy may also cause pollution to water resources. Thus, it is extremely urgent to take effective approaches
to improve the waterbodies. In this study, four novel denitrifying Bacillus strains, JD-005, JD-014, JD-016 and
JD-017, were isolated, characterized and further studied on the properties of nitrogen removal. The four strains
could utilize ammonium, nitrate and nitrite source under aerobic conditions. In a simulated 5 L sewage biore
actor, four strains also exhibited efficient denitrification performance in multiple nitrogen coexistence, with the
nitrite-N removal rates reached 0.41–1.89 mg/L/h, and the ammonium removal rates reached 0.87–2.07 mg/L/
h. In addition, the denitrification metabolic pathway of Bacillus subtilis JD-014 was confirmed by investigating
the nitrogen balance, gaseous nitrogen production, enzyme activity and functional genes analysis. With
outstanding nitrite tolerance, the nitrogen removal efficiency could up to approximately 50–80% at initial NO2 -
N concentration of 50–200 mg/L. These results showed that JD-014 should be of great potential in bioremedi
ation of polluted waterbodies and may provide an efficient and eco-friendly way in the future to improve water
resources and realize better sustainable bioenergy application.
1. Introduction resources for energy generation. However, most crops for biofuels have
high irrigation water requirements and compete for already scarce
Energy shortage and environmental pollution have become the water. For example, ethanol processing requires more water per unit of
constraint of economic development in many countries. The satisfaction biofuel produced than petroleum refining [8]. It is reported that a liter of
of vast demand of global energy and the per capita energy usage is the ethanol made from sugarcane in India needs 3500 L of water, while in
current key point of research [1]. Among the different available China it takes nearly 2400 L of irrigation water when using maize as raw
renewable energy sources, bioenergy from the use of biomass is material [9]. What’s more, to meet the demands of mass production and
considered as one of the most potential options worldwide to meet the increase crop yields, large-scale land would change to produce bio
energy demands and ensure energy security, due to its easy availability, energy crops and the application of extra chemicals and fertilizers is
widely distribution and inexpensive nature [2]. Although it usually inevitably increased. This will undoubtedly impair water quality, lead
thought of as harmless, this does not mean that it will be no impact on ing to an accumulation of nitrogen and toxic substances in waterbodies
the environment. In fact, it exists at the expense of hidden environ [10–12]. In addition, industrial-scale production of bioenergy may also
mental burdens, such as putting great pressure on water consumption cause pollution to water resources. For example, the major challenge of
and water quality [3–5]. large scale micro-algae biofuel production is the enrichment and
Nowadays, there is a move towards planting of perennial bioenergy leaching of residual nutrients, such as nitrogen and phosphorus, that
crops to help reduce the energy shortage around the world, especially in increased the risk of water contamination [13]. Thus, it is extremely
some low- and middle-income countries, such as India, Pakistan, urgent to take effective approaches to improve the water quality. While
Malaysia and China [1,6,7]. These countries are always rich in biomass in water bodies, nitrogen contamination has been considered as the most
* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (Y. Xin), [email protected] (L. Zhang).
https://doi.org/10.1016/j.biombioe.2020.105677
Received 23 July 2019; Received in revised form 31 May 2020; Accepted 28 June 2020
Available online 17 July 2020
0961-9534/© 2020 Elsevier Ltd. All rights reserved.
T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
prevalent and excessive one, which could lead to eutrophication, algal Nitrification medium (NM) was used for the determination the
bloom, impair aquatic habitat of surface waterbodies and further threat ammonium removal ability of isolated strains, contained (per liter):
the safety of public health [14,15]. Hence, the removal of nitrogen (NH4)2SO4 0.71 g, Na2HPO4 4.0 g, KH2PO4 1.5 g, MgSO4⋅7H2O 0.2 g,
compounds is the key point in water management. In response, more sodium succinate 8.5 g, trace element solution 2 mL, pH 7.0–7.5.
attention is focused on the biological nitrogen removal technologies. The trace element solution (per liter) was composed of EDTA 50 g,
Comparing with other traditional denitrogenation approaches, they ZnSO4⋅7H2O 3.92 g, CaCl2 5.5 g, MnCl2⋅4H2O 5.06 g, FeSO4⋅7H2O 5.0 g,
offer the advantages of high efficiency, low cost and do not create sec (NH4)6Mo7O2⋅4H2O 1.1 g, CuSO4⋅5H2O 1.57 g and CoCl2⋅6H2O 1.61 g.
ondary pollution or residues [16].
Generally speaking, a conventional biological treatment process 2.2. Isolation and identification of aerobic denitrifying Bacillus strains
often includes autotrophic nitrification, in which nitrifiers convert
ammonium to nitrite or nitrate under aerobic conditions, and anaerobic Samples were collected from different environments such as forests,
denitrification, in which denitrifiers reduce nitrate and nitrite to fish, shrimp and crab culture ponds, activated sludge and sediments. 2
nitrogenous gas [17]. However, the application of this traditional pro mL water samples or 2 g sediments were transferred into 250 mL ster
cess is limited due to the separation of aerobic and anoxic phases, ilized conical flasks containing 98 mL liquid LB medium and the flasks
time-consuming, cost-intensive and different demands of oxygen and were put into a shaking incubator which kept at 37 � C, 200 rpm. 0.1 mL
organic substrates [18]. Consequently, there is still a great demand to of gradient dilutions were then spread on LB plates. Separate colonies
find new techniques to remove nitrogen compounds during wastewater were picked out and purified by repeated streaking on agar plates. Then,
treatment [19–21]. Aerobic denitrification, a new biological process, is the isolated were transferred and dropped into BTB medium plates at 30
being investigated recently. Microorganisms with this ability can �
C for 3 days. When the BTB medium turned blue, the colonies were
simultaneously conduct nitrification and denitrification in one reactor inoculated to 100 mL DM to test the denitrification abilities. The strains
under identical overall operating conditions and exhibit much higher with the highest denitrification rate were selected and stored in 30%
growth rate, so that the whole process could be achieved for simple glycerol solution at 80 � C for further analysis. The Gram staining re
process, high efficiency, low investment and less operation cost [18,20]. action was determined using the method described by Gerhardt et al.
As this new biological nitrogen removal technology is of major [29] and the morphology of selected strains were taken with a scanning
importance to the removal of nitrogen pollution, studies of microbes electron microscope (SU8220, Hitachi, Japan).
that have this function are needed to solve the problem of eutrophica To identify the isolated strains, the genomic DNA of the strains were
tion in water bodies and maintain the nitrogen balance of ecosystems. extracted from bacterial suspensions in LB medium with Biospin bac
Recent research has focused mainly on the screening of microbes with teria genomic DNA extraction kit (BioFlux, Shanghai, China), according
high nitrogen-removing efficiencies [22,23]. Thus far, a large number of to the manufacturer’s instructions. The genomic DNA was used as a
these functional strains, mainly Gram-negative bacteria, such as Pseu template to amplify 16S rRNA gene by PCR using bacterial universal
domonas sp. [24], Alcaligenes sp. [14], Paracoccus sp. [25] and Acineto primers 27F and 1492R. After purification, PCR-amplified product was
bacter sp. [18], have been reported. Meanwhile, Bacillus genera, which sequenced by Sangon Biotech (Shanghai, China) Co. Ltd. The 16S rRNA
are typical Gram-positive probiotics, popularly used in the aquaculture sequences were compared with that of other available microorganisms
industry due to their biosecurity, broad environmental adaptation, in GenBank of NCBI by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/
improvement of water quality and promotion of the growth of aquatic Blast.cgi). A phylogenetic tree was constructed using the neighbor
animals [26–28]. But compared with other strains, the aerobic deni joining method by MEGA 5.2 software.
trifying characteristics and the nitrogen removal mechanism of
wild-type Bacillus strains are still poorly understood. 2.3. Estimation of nitrogen removal capability of Bacillus strains
Therefore, four aerobic denitrifying Bacillus strains were isolated and
identified from different environmental samples in our study. Their ni The screened strains were cultivated for 16 h at 37 � C, 200 rpm in LB
trogen removal characteristics were investigated using different nitro medium, and the bacterial suspensions were collected by centrifugation
gen sources. To gain valuable insight into the metabolic pathway of at 5000 rpm for 10 min and washed with sterile water for three times.
denitrification in Bacillus strains, nitrogen balance, gaseous nitrogen Then, 1 mL bacterial suspensions were placed into 100 mL sterile DM or
production and the analysis of the functional genes were studied in JD- NM to keep the initial OD600 at approximately 0.1. The conical flasks
014. This study may provide an optional and safe microbial resource in were incubated at 37 � C, 200 rpm for 5 days. During the fermentation
the field of green nitrogen removal and will be beneficial for improving period, samples were taken periodically to determine the cell density
water resources utilization and enhancing the performance of bioenergy (OD600). Then centrifuged at 12,000 rpm for 10 min. The supernatant
application. was used for analysis of the pH, NO3 -N, NO2 -N, NHþ 4 -N, NH2OH and TN.
All the experiments were performed in triplicate.
2. Materials and methods
2.4. Detection of gaseous nitrogen
2.1. Media
The culture suspensions were inoculated into 100 mL sterile DM or
Luria Bertani (LB) medium was used for the enrichment and isolation NM with different nitrogen sources and sealed in 250 mL serum bottles.
of denitrifying bacteria, contained (per liter): peptone 10 g, yeast extract To simulate the amount of oxygen in the air, the bottles were evacuated
5 g, NaCl 10 g. For LB plate, 20 g agar was added. and fully aerated with O2–He (21:79) gas. Then the bottles were culti
Bromothymol Blue (BTB) medium was used for the preliminary vated at 37 � C and 200 rpm for 5 days. Gas samples from the headspace
screening of denitrifying bacteria, contained (per liter): NaNO3 0.84 g, were collected by gas tight syringes to detect N2 and N2O using gas
KH2PO4 1.0 g, FeSO4⋅7H2O 0.59 g, CaCl2 0.09 g, MgSO4⋅7H2O 1.0 g, chromatography. The bottle without inoculation was used as a control.
sodium succinate 8.5 g, BTB (1% alcoholic solution) 1 mL, agar 20 g, pH Cultures were sampled for chemical analysis.
7.0–7.3.
Denitrification medium (DM) was used for the determination the 2.5. Amplification of functional denitrification genes
nitrate or nitrite removal ability of isolated strains, contained (per liter):
NaNO3 0.84 g or NaNO2 0.74 g, Na2HPO4 4.0 g, KH2PO4 1.5 g, The genomic DNA of the isolated strains were used as templates to
MgSO4⋅7H2O 0.2 g, sodium succinate 8.5 g, trace element solution 2 mL, amplify the fragments of denitrification functional genes nap, nor and
pH 7.0–7.5. nos. The primers used in the study were listed in Table 1. The PCR
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T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
products were separated by 1.5% agarose gel electrophoresis. Bands removal rate was: RRðmg =L =hÞ ¼ T1 T T2 . Where T1 and T2 represent for
were photographed using the gel imaging system (ChemiDoc XRSþ, Bio- the initial concentration of nitrogen and nitrogen concentration at time
Rad, USA). T, respectively.
B.subtilis JD-014 was harvested after cultivation for 60 h in DM by 3.1. Isolation and identification of the denitrifying strains
centrifugation at 4 � C, 12,000 rpm for 10 min, washed three times and
then resuspended in 10 mmol/L phosphate buffer (pH 7.4). Cell-free A total of 21 preliminary selected strains from BTB plates were
extracts were obtained by subjecting the bacterial suspensions to lysis inoculated in DM to test the denitrification capability. Four Bacillus
using ultrasonication and then centrifuged at 12,000 rpm for 10 min. strains, named as JD-005, JD-014, JD-016 and JD-017 showed higher
The activity of nitrate reductase was determined by the reduction of nitrogen removal rate and were chosen for further analysis. Homology
nitrate with NADH as the electron acceptor [30]. Protein concentration searches of the 16S rRNA gene sequences revealed that the four strains
in the cell-free extract was determined by Bradford reagent kit (Sangon, were most closely related to Bacillus pumilus, Bacillus subtilis, Bacillus
Shanghai, China). One unit of enzyme activity (U) was defined as the licheniformis and Bacillus subtilis, respectively, and the phylogenetic tree
amount of enzyme that catalyzed the conversion of 1 μmol of substrate (Fig. 1) was constructed based on 16S rRNA sequence. The nucleotide
per minute. The specific activity (U/mg) was defined as the amount of sequences were submitted to GenBank under the accession numbers
enzyme units divided by the total amount of protein in mg. MK124647 to MK124650.
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T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
Fig. 1. Phylogenetic tree constructed from 16S rRNA gene sequences of strains JD-005, JD-014, JD-016, JD-017 and comparative reference strains by the neighbor-
joining method. Bar, 0.01 denotes the genetic distance.
Fig. 2. Changes in various substances during fermentation when NO3 -N was the sole nitrogen source. (a) OD600 and pH, (b) NO3 -N, (c) NO2 -N, (d) NHþ
4 -N.
trace concentration of NO2 -N was detected during the entire process 3.2.4. Nitrogen removal characteristics in synthetic wastewater of Bacillus
(Fig. 4c–d). strains
A mixed nitrogen source (50 mg/L NHþ 4 -N and NO2 -N) was used in a
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T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
Table 2
Comparison of nitrification rates and denitrification rates of Bacillus strains.
Strains Initial nitrifier Nitrification Initial Denitrification Operation Culture Media References
concentration rate denitrifier rate conditions
Carbon Nitrogen source C/N
concentration
source ratio
simulated 5 L sewage bioreactor to elucidate the aerobic nitrogen 69.69% and 77.73%, with the maximum ammonium removal rates were
removing of the four selected Bacillus strains in large-scale application. 0.87, 1.86, 2.07 and 1.35 mg/L/h, respectively, while the nitrite
The results in Fig. 5 demonstrate that JD-005 grew much slowly and the removal efficiency were 74.71%, 99.97%, 99.97% and 56.75%, with the
OD600 reached a peak value of 2.05 at 72 h, while the biomass of the maximum nitrite removal rates were 0.55, 1.89, 1.56 and 0.41 mg/L/h,
other three strains gained the maximum at 28–36 h. For nitrogen respectively. The results indicate that the strains can significantly alle
removal, the isolated strains JD-005, JD-016 and JD-017 preferred to viate the pollutant contents in wastewater with multiple nitrogen
remove ammonium in the mixed N-source, while the strain JD-014 coexistence.
reduced the ammonium nitrogen and nitrite nitrogen nearly at the
same time. Moreover, the ammonium removal efficiency of the four
strains JD-005, JD-014, JD-016 and JD-017 were 73.15%, 69.81%,
5
T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
Fig. 3. Changes in various substances during fermentation when NO2 -N was the sole nitrogen source. (a) OD600 and pH, (b) NO2 -N, (c) NO3 -N, (d) NHþ
4 -N.
NO2 -N.
3.3. Nitrogen balance analysis and gaseous nitrogen detection nitrogen removal process was calculated. As depicted in Table 3, by
comparing the initial and final N mass of Bacillus JD-014 in the reaction
To study the nitrogen transformation of JD-014, the gaseous nitrogen system, 13.72–22.60% of the initial nitrogen was changed to intracel
in the headspace were monitored and the nitrogen balance during the lular N when NHþ4 -N, NO2 -N, or NO3 -N were used as the sole nitrogen
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Fig. 5. Simultaneous nitrification and denitrification characteristics of the four isolated Bacillus strains with mix N-sources in a 5 L bioreactor system. (a) Strain JD-
005, (b) Strain JD-014, (c) Strain JD-016, (d) Strain JD-017.
Table 3
Nitrogen balance of the selected strain JD-014 (unit: mg/L).
N sources NHþ
4 -N NO3 -N NO2 -N Intracellular N N2O N2
NHþ
4 -N Initial 48.17 � 1.07 – – – – –
Final 15.83 � 2.42 0.71 � 0.03 0.01 � 0 10.89 � 2.68 0.0022 � 0.0002 20.78 � 3.53
NO¡
3 -N Initial 0.22 � 0.01 62.89 � 0.48 – – – –
Final 24.60 � 1.32 10.36 � 1.65 0.77 � 0.20 10.99 � 1.29 0.0049 � 0.0003 16.39 � 1.33
NO¡
2 -N Initial 0.92 � 0.08 16.28 � 0.10 56.12 � 0.73 – – –
Final 30.76 � 1.02 0.07 � 0 4.24 � 0.16 10.06 � 0.87 0.0002 � 0 29.31 � 1.74
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with the escalating scale of biomass crops cultivation, large water de
mand and water quality destruction are two important factors affecting
environmental sustainability [1,4]. While in recent years, more atten
tion has been paid to achieve the sustainable development of bioenergy,
the environmental aspects should also be taken into consideration [34].
Thus, it is of great significance to take the appropriate technologies to
improve waterbodies.
Water treatment in low- and middle-income countries is facing great
problems, such as high cost, the demand of special skills and profes
sional equipment [35]. Biotechnology is the most common, efficient,
and cost-effective method in solving the problem of water pollution for
those countries, and the discovery of aerobic denitrifying bacteria opens
a new way for biological denitrogenation technology. The above pre
sented denitrifying Bacillus strains with promising nitrogen removal
performance, which could be well applied to the purification of inten
sive polluted water. The treatment of these strains could be achieved for
simple operation, low costs, eco-friendly and to some degree, it will not
put additional burden on the economy for the low- and middle-income
countries.
To understand the denitrification characteristics of the four selected
Bacillus strains, nitrate (NO3 -N) and nitrite (NO2 -N) were separately
Fig. 7. PCR amplification results of functional genes from Bacillus JD-014. M: used as the sole nitrogen source under aerobic culture conditions. The
DL10000 DNA Maker; lane 1: The PCR products of nap gene (2040 bp); lane 2:
accumulation of ammonium was observed during the degradation of
The PCR products of nor gene (1917 bp); lane 3: The PCR products of nos gene
nitrate or nitrite, which might be attributed to the autolysis of some cells
(1755 bp).
during the later stage of fermentation, so that the intracellular ammo
nium nitrogen could release into the medium. In addition, the organic
mU/mg protein, when the strain JD-014 was cultivated in DM for 60 h.
nitrogen of death cells could also convert to ammonium nitrogen.
The transcriptional analysis of key genes involved in the denitrifi
Similar results were also reported in previous studies [17,36].
cation pathway of B. subtilis JD-014 was carried out by qRT-PCR
As is well-known, nitrite has a significant toxicity that is harmful to
(Fig. 8a). With the rapidly degradation of nitrate (Fig. 8b), the expres
aquatic animals, leading to a large variety of physiological disturbances
sion level of nap gene decreased compared with that at the first day.
and immunosuppression [37,38]. When NO2 -N was used as the sole
While expression of nor and nos gene significantly increased by 2.73 and
nitrogen source, it took a long time for the four selected Bacillus strains
2.85 times at the 3rd day, respectively, which corresponded to the
to adapt to such a stressful environment owing to the toxic effect of
exponential stage in growth curve of JD-014 (Fig. 8b).
nitrite on the cells. Due to the oxidization of NO2 -N, a certain amount of
To further investigate potential inducible mechanism of nitrogen
NO3 -N (approximately 35 mg/L) was detected at the beginning, but
removing associated genes, the effect of initial nitrate concentration on
then it decreased because of the utilization by the strains. The same
the removing of NO3 -N was evaluated under aerobic condition (Fig. 9).
phenomenon was also found in the study of Klebsiella sp. TN-10 [39],
As initial NO3 -N concentration increased from 10 to 300 mg/L, the
which detected 15.43 mg/L NO3 -N when NO2 -N was added. Addition
OD600 value gradually increased from 0.70 to 3.44, while the NO3 -N
ally, it is reported that a high nitrite content strongly inhibited denitri
removing also increased; The specific NO3 -N degradation per OD600 at
fication activity and a nitrite concentration of 20 mg/L was the critical
NO3 -N concentration 10, 50, 100, 200, and 300 mg/L was 22.95, 49.30,
value for bacterial denitrification [38]. The results of our study indicate
49.82, 55.81 and 75.02 mg/L, respectively. These results indicate that
that the selected strain JD-014 exhibit strong nitrite toxicity tolerance
the gene expression in the denitrification should be inducibly controlled
and have a good prospect in widely used in high-nitrite wastewater
and the expression level varied with the constant change of initial nitrate
treatment. The tolerance is much higher than that of another aerobic
concentration.
denitrifier, Bacillus coagulans YX-6, which maintained highly effective
nitrite removing compared with other Bacillus strains [38]. It was re
4. Discussion
ported that the NO2 -N degradation efficiency of YX-6 could almost
reach 100% at the 20 mg/L initial concentration, and it declined grad
Biomass is an emerging renewable source for production of bio
ually to only 20% with the increase of NO2 -N concentration to 100
energy all over the world to alleviate the energy crisis. However, along
mg/L. While the nitrogen removal efficiency of JD-014 could be up to
Fig. 8. Relative expression levels of genes and denitrification characteristics of B. subtilis JD-014.
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T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
Fig. 9. The growth (a) and nitrate removal characteristics (b) of Bacillus. JD-014 under different initial NO3 -N concentrations.
approximately 50–80%, at 50–200 mg/L initial NO2 -N level. essential enzymes [47], including nitrate reductase (Nar or Nap), nitrite
The nitrogen removal rates of the isolated strains in this study were reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase
compared with those reported for other Bacillus genera. We summarized (Nos). However, some isolated strains have exhibited nitrogen removal
the relevant studies on nitrogen removal rates of Bacillus spp. in Table 2 abilities via an incomplete denitrification process, meaning that at least
and found that the nitrate removal rates of the isolated strains in this one reductase-encoding gene cluster is missing [48,49]. NO3 -N is con
study were lower than that of B. cereus GS-5 [22] with nitrate removal verted to NO2 -N by the nitrate reductase Nar or Nap as the first step in
rate of 2.69 mg/L/h, but higher than that of B. subtilis BRAZ2B (0.53 this pathway. Nar locates on the cytoplasmic membrane and is expressed
mg/L/h) [40], B. litoralis N31 (0.59 mg/L/h) [17], Bacillus sp. LY (0.35 under anaerobic conditions, whereas Nap, which locates on the peri
mg/L/h) [41] and B. cereus X7 (0.32 mg/L/h) [42]. The nitrite removal plasmic compartment, is insensitive to O2 and the presence of Nap would
rates for each strain were comparable or even higher than those of the be responsible for the aerobic conversion of nitrate to nitrite [50]. In our
functional Bacillus reported previously (Table 2), such as B. coagulans study, the functional nap gene was amplified successfully in Bacillus
YX-6 (0.71 mg/L/h) with initial nitrite 10 mg/L [38], B. cereus GS-5 JD-014 and the nitrate reductase was also successfully expressed in the
(1.18 mg/L/h) with initial nitrite 50 mg/L [22], B. megaterium S379 cell free extracts.
(0.50 mg/L/h) with initial nitrite 65 mg/L [22], B. cereus PB88 (0.25 For the reaction NO2 -N to NO, catalyzed by nitrite reductase. Ac
mg/L/h) with initial nitrite 12 mg/L [43], and Bacillus sp. LY (0.44 cording to the different structures and catalytic sites, the nitrite reduc
mg/L/h) with initial nitrite 20 mg/L [41]. tase can be divided into a copper type enzyme and a cytochrome cd1-
When NHþ 4 -N was used as the sole nitrogen source under aerobic type enzyme expressed by nirK and nirS, respectively. These two types of
culture conditions, the maximum nitrogen removal rates were nitrite reductase usually cannot present simultaneously in the same
0.86,1.67, 0.70 and 0.71 mg/h/L for each strain, respectively, which strain [51]. Unfortunately, both the nirK and nirS were not amplified.
were higher than that of Bacillus sp. LY (0.42 mg/L/h) when the initial Recently, some new findings have demonstrated that it is insufficient to
ammonium was 42.5 mg/L [41] and Bacillus cereus X7 (0.77 mg/L/h) gauge denitrification potential, or to predict the production of gaseous
when the initial ammonium was 50 mg/L [42]. NH2OH, one of the nitrogen, just through the monitor of the nirK and nirS genes. Onley et al.
significant intermediates in the process of ammonium removal, was [52] reported that a common soil bacterium Anaeromyxobacter dehalo
hardly detectable in the medium because of its instability and rapid genans lacked the key denitrification genes nirS and nirK, but still
conversion to other downstream intermediates [44]. There was only exhibited nitrogen removal ability with the help of iron. Mauffrey et al.
trace concentration of NO2 -N detected during the entire process. The [49] isolated a methylotrophic marine bacterium JAM1 from a deni
main reason may be that the processes of nitrification and denitrification trification reactor, which was identified as Methylophaga nitratir
were in a balanced state, so that nitrite did not accumulate in the cul educenticrescens. It could still produce nitrogenous gases such as NO and
tures. This observation was consistent with previous studies on Bacillus N2O under anaerobic conditions, despite the absence of the genes nirS
litoralis N31 [17], Klebsiella pneumoniae CF-S9 [45] and Acinetobacter sp. and nirK. There may exist a new type of nitrite reductase in strain
Y16 [20]. JD-014, which needs in depth study.
To better understand of the nitrogen removal characteristics of the In addition, the expression levels of these key genes involved in the
selected strains, Bacillus subtilis JD-014, which showed the most efficient denitrification pathway of B. subtilis JD-014 were investigated under
heterotrophic nitrification and aerobic denitrification with nitrite different fermentation phases and a similar trend was achieved by
compared with the other Bacillus strains, was chosen to monitor the gas Pseudomonas sp. JQ-H3 [53], showing that high expression levels of nor
nitrogen products and nitrogen balance was also calculated. N2 and N2O and nos could ensure sufficient production of NO or N2O reductase to
were indeed detected in our closed system. Aboobakar et al. [46] re maintain low levels of NO and facilitate the reduction of N2O to N2.
ported that the presence of oxygen can inhibit nitrous oxide reductase Thus, these results provide additional evidence that the nitrogen
during the denitrification process, leading to large amounts of N2O removal pathway of the isolated B. subtilis JD-014 was activated through
generated as the end product instead of N2. While in our study, N2O, a aerobic denitrification.
greenhouse gaseous denitrification product, was produced in a very
small proportion. Taken together, JD-014 indeed convert part of nitro 5. Conclusions
gen compounds to gaseous products through aerobic denitrification
pathway and have great advantage in practical application of biological In order for biomass to be a better alternative to conventional fuels,
nitrogen removal with negligible emission of nitrous oxide. negative environmental impacts on water quality and the resulting
To further investigate the denitrification metabolic pathway of JD- economic costs from the biomass production should be reduced as much
014, the denitrification functional genes were analyzed. Generally as possible. Aerobic denitrification is considered as one of the most
speaking, the complete denitrification process is catalyzed by four efficient, and cost-effective biotechnologies to improve waterbodies. In
9
T. Yang et al. Biomass and Bioenergy 140 (2020) 105677
this study, four aerobic Bacillus denitrifying strains were isolated and application for the treatment of high-strength nitrogenous wastewater, Bioresour.
Technol. 193 (2015) 227–233.
characterized. All the selected strains presented great capabilities to
[19] P. Chen, J. Li, Q.X. Li, Y. Wang, S. Li, T. Ren, L. Wang, Simultaneous heterotrophic
reduce nitrate, nitrite and ammonium under aerobic conditions. It was nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24,
found that strain JD-014 exhibited the most efficient aerobic denitrifi Bioresour. Technol. 116 (2012) 266–270.
cation, with little greenhouse gas nitrous oxide emission. Moreover, JD- [20] X. Huang, W. Li, D. Zhang, W. Qin, Ammonium removal by a novel oligotrophic
Acinetobacter sp. Y16 capable of heterotrophic nitrification-aerobic denitrification
014 performed strong resistance to high toxic concentration of nitrite, at low temperature, Bioresour. Technol. 146 (2013) 44–50.
the removal efficiency could still up to approximately 50% at an initial [21] Q. Liang, X. Zhang, K.H. Lee, Y. Wang, K. Yu, W. Shen, L. Fu, M. Shu, W. Li,
NO2 -N concentration of 200 mg/L, suggesting a great potential for Nitrogen removal and water microbiota in grass carp culture following
supplementation with Bacillus licheniformis BSK-4, World J. Microbiol. Biotechnol.
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