Ecotoxicology and Environmental Safety 218 (2021) 112287

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Development of the yeast and lactic acid bacteria co-culture agent for
atmospheric ammonia removing: Genomic features and on-site applications
Yanfang Zhang a, e, Zhimin Dai b, f, Zhicheng Zhou c, Huaqun Yin a, e, Min Zhang a, e,
Hetian Zhang a, e, Yongjun Liu c, Qian Li a, e, Xiaolong Nan d, Xueduan Liu a, e, Delong Meng a, e, *
a
School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China
b
Central South Water Science and Technology Co. Ltd, Changsha 410001, China
c
Hunan Tobacco Science Institute, Changsha 410010, China
d
306 Bridge of Hunan Nuclear Geology, Changsha 410083, China
e
Key Laboratory of Biometallurgy, Ministry of Education, Central South University, Changsha 410083, China
f
National City Water Supply Water Quality Monitoring Network Changsha Monitoring Station, Changsha 410001, China

A R T I C L E I N F O A B S T R A C T

Edited by Dr. Yong Liang Odorous gas (e.g. atmospheric ammonia) in low ventilation public places, such as public toilets and waste
transfer stations, causes severe health problems. Many technologies are developed to purify the atmospheric
Keywords: ammonia, among which the microbial agents are supposed to be a green and economical approach. In this study,
Atmospheric ammonia we developed a yeast, Pichia sp. J1, and a lactic acid bacterium (LAB), Lactobacillus paracasei B1, co-culture agent
Bio-purification
for atmospheric ammonia removing. The on-site application results indicated the yeast and LAB mixed fermented
Yeast and lactic acid bacteria co-culture agents
agent had a maximum ammonia removing efficiency of 98.78%, which is significantly higher than the pure
On-site application
Genomic features cultures (78.93% for B1 and 75.00% for J1), indicating the co-culture agent is an excellent biological product for
ammonia removal. The excellent performance of the agent is closely related to the synergy behaviors between
the yeast and LAB. In the co-culture agents, some of the LAB cells adhered closely to the yeast, and the growth
and lactic acid producing ability of LAB were significantly promoted by yeast. Genomic analysis indicated the
complementary of nutrients, i.e. carbon and nitrogen resources, signal transduction, and adhesion proteins
(regulates adhesion behavior) played roles in regulating the synergy effects. Our study offers a novel biological
solution of odorous gas purification.

1. Introduction
Poor air quality is the 9th largest global burden of disease risk
(Forouzanfar et al., 2015). Odorous gas is a common air pollution
existing in public toilets, livestock industry, and organic solid waste
treatment (composting) (Hafner et al., 2013; Zong et al., 2015; Kang
et al., 2020). Ammonia (NH3), the product of biological degradation of
protein, urea, and other nitrogen components, is one of the main com­
ponents of odorous gas. In low ventilation conditions, the ammonia
concentration can be up to 200 ppm (Wheeler et al., 2006). Air ammonia
causes severe health problems for humans and animals (Donham, 1991),
by stimulating mucosa, and the respiratory tract.
Many technologies are developed to remove NH3 from the air,
including conventional combustion, neutralization, oxidation, adsorp­
tion, absorption, and biological method (Ashtari et al., 2016; Sheng
et al., 2020). Treating air ammonia with biological agents is a green,
economical and efficient approach. Microorganisms have demonstrated
to have a good ability to remove ammonia in many environments, such
as wastewater or solid wastes. For example, nitrification and denitrifi­
cation bacteria are often used for treating ammonia-rich wastewater
(Sliekers et al., 2002; Peng and Zhu, 2006). The ammonia oxidizers can
utilize NH3 as nutrients or energy substance and oxidize the NH3 to NO-3
(Martens-Habbena et al., 2009), most of the microorganisms can
assimilate NH3 to form L-glutamine (Pengpeng and Tan, 2013), and acid
producing microbes can produce acids to neutralize NH3. Therefore, the
microbial agents are supposed to have good ammonia removal ability in
the air. However, using only microbes (not combined with biofilter) to
purify air NH3 is seldom reported.
Bacteria and fungi co-cultivated agents are widely used in many
aspects, such as wine production (Alexandre et al., 2004), waste

* Correspondence to: Central South University, No. 932 Lushan South Street, Yuelu District, Changsha 410083, China.
E-mail address: delong.meng@csu.edu.cn (D. Meng).

https://doi.org/10.1016/j.ecoenv.2021.112287
Received 4 January 2021; Received in revised form 22 March 2021; Accepted 22 April 2021
Available online 29 April 2021
0147-6513/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Zhang et al.
treatment (Zhao et al., 2017), volatile organic compounds purification
(Cheng et al., 2016), food industry (Sieuwerts et al., 2010) and etc., as
the fungi often promote the growth and interesting function of the
bacteria. Lactic acid bacteria (LAB) produce lactic acid and use ammonia
as a nitrogen source, thus, we speculated that the LAB agent could purify
air ammonia. The LAB is often found to coexist with yeast in the
ecosystem, and share the same environment (Maligoy et al., 2008). The
combination of yeast and LAB was successfully applied in food and dairy
fermentation (Dequin and Barre, 1994; Sieuwerts et al., 2010).
Furthermore, not only the yeast fungi promote the LAB growth and
lactic acid production, but they also showed excellent ammonia assim­
ilation ability (Grenson et al., 1974; Magasanik, 2003). Therefore,
although the yeast and LAB coculture is seldom reported to be used in
environmental bioremediation, particularly the air purifying, the mi­
crobial flora containing yeast and LAB have great potential in atmo­
spheric ammonia removal.
The interaction between yeast and LAB is well studied. The yeast and
LAB cooperated in carbon resources, purine, amino acid and long-chain
fatty acid metabolism, iron uptake, and environment modification
(Maligoy et al., 2008; Mendes et al., 2013). However, the signal trans­
duction between yeast and LAB was still unclear. Quorum sensing (QS)
is first referred to the intercellular (cell-to-cell) signal exchange between
bacteria, to adapt to environmental stress in time and space (Winzer
et al., 2002; Hense et al., 2007), and it has been expanded to other
microorganisms such as yeasts (Sprague and Winans, 2006; Brexo and
Sant’Ana, 2018). Quorum sensing is conducted through small and
diffuse molecules, that are called auto-inducer molecules (AI). It is
known that AIs are closely associated with the improvements of mi­
croorganisms to assess nutrients (Skandamis and Nychas, 2012). It is
hypothesized that the QS is a beneficial tool to maintain the ecological
stability in yeast and bacteria (e.g. LAB) fermentation system (Brexo and
Sant’Ana, 2018; Almeida et al., 2020). However, the QS system in yeast
and LAB cocultured fermentation vat is unclear, and it still lacks
experimental evidence.
In this study, we isolated and selected two microbial strains, a lactic
acid producing bacterium and a yeast fungus, that could purify atmo­
spheric NH3 from high ammonia (up to 75 mg/m3) environments and
had excellent industrial application effects. Interestingly, the co-culture
agent of the two strains had much better than the pure cultures. The
genomic analyses were carried out to reveal the atmospheric NH3
removing mechanisms and the mutual enhancement mechanism of the
two microorganisms. Our results provided a new approach for purifying
odorous ammonia in public places, and a novel application approach of
yeast and lactic acid bacteria cocultures.
2. Materials and methods
2.1. Enrichment and isolation of ammonia-removing microorganisms
The ammonia removing microorganisms (ARMs) were isolated from
the waste leachate. The leachate samples were collected from the waste
transfer station (E 28.1736, E112.9288) in Changsha City, China. The
Luria-Bertani broth (LB) and yeast extract-peptone dextrose broth (YPD)
medium (Table S1) were used to enrich microorganisms from leachate.
To isolate ARMs, the enrichments were inoculated to ARM selective
medium and incubated for 72 h at 30 ℃ and 150 rpm. The ARM selective
medium contained 36 g/L of sucrose, 2 g/L of KH2PO4, 0.5 g/L of
MgSO4, 2.0 g/L of NaCl, 0.1 g/L of ZnSO4 and 2.5 g/L ammonia, the
medium pH was adjusted to 7.0. One bacteria strain B1 and one fungal
strain J1 with high removal efficiency were isolated and used for further
experiments (Fig. S1).
2.2. Identification of ammonia-removing microorganisms
Genomic DNA of bacterial and fungal pure culture was extracted
using the TIANamp Bacteria DNA Kit (TIANGEN, China) and the fungal
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Ecotoxicology and Environmental Safety 218 (2021) 112287
DNA kit (Omega), respectively, following the manufacture’s manual.
The 16S rDNA sequence was amplified by the 27F（5′ -AGAGTTTG
ATCCTGGCTCAG-3′ ）and 1492R（5′ -TACGGYTACCTTGTTACGACTT-
3′ ）primer pair, and the fungal ITS sequence was amplified by the ITS1
(5′ -TCCGTAGGTGAACCTGCGG-3′ ) and ITS4 (5′ -TCCTCCGCTTATTGA­
TATGC-3′ ) primer pair. PCR was performed on a Thermal Cycler 2720
(Applied Biosystems, Foster City, CA, USA), and PCR conditions were list
in Table S2. The PCR products were sent to Shenggong company
(Shanghai, China) for sequencing. Sequences were blasted to NCBI for
identification of ARMs. Operational details of bacterial identification
can refer to the literatures (Wu et al., 2020; Lin et al., 2011).
2.3. Preparation of ammonia removing agents
Two ARMs, a bacterium and a fungus, were fermented in pure cul­
ture and co-culture systems, respectively, resulted in three biological
agents, namely bacterial B1 pure culture, fungal J1 pure culture and co-
culture agents. The cultivation and fermentation medium were the same
for both bacterial and fungal ARMs, with the contents of 0.3 g/L glucose,
0.2 g/L yeast extract powder, 0.01 g/L KH2PO4 and 0.01 g/L MgSO4. The
ARMs were firstly cultivated in an Incubator Shaker (ZHWY – 2112B,
ZHICHENG, Shanghai) at 160 rpm, 30 ℃ and subsequently, the ARMs
were inoculated to a 5 L tank reactor (BTF-A5L, Bio-Top Inc, Taichung,
Taiwan) for fermentation. The ARMs were fermented for 72 h and the
total fermentation volume was 3 L with the inoculation of 10% (V/V)
cultivated ARMs. The ARMs were fermented at 30 ℃ with the aeration
rate of 1.0 vvm (volume air/volume liquid/min), and the agitation rate
of 50–150 rpm to maintain the dissolved oxygen (DO) concentration at
60–100%. The pH was not controlled. After 24 h of fermentation, the
glucose solution was fed into the fermenter to maintain the glucose
concentration of 5–10 g/L. For the co-culture fermentation system, the
bacterium was firstly fermented for 24 h and then the fungus was
inoculated to the bacterial fermentation system with the inoculation
concentration of 0.5% (v/v). The pH, cell density and DO were recorded
during the whole fermentation stage. The pH and DO were determined
by the sensors on the fermentation reactor, the cell density was deter­
mined by the cell count method with a hemocytometer under a micro­
scope (BX41, Olympus, Tokyo, Japan). Microbial cells were observed
under 1000× microscopes after they were stained by carbol fuchsin for
2 min
The fermentation products were diluted with distilled water to the
final cell density of 1 × 108 cfu/ml (colony forming units/ml). The
diluted fermentation products were used as ammonia removal biological
agents.
2.4. Application of ammonia removing agents at public places
Three agents (B1 pure culture, J1 pure culture and co-culture
agents), together with the water (as control) were used to test the
ammonia removing efficiency of the agents. The NH3 removal experi­
ments were carried out at two public places, the waste transfer station
(WTS) of Shuda College and the public toilet (PT) of Huanghe Com­
munity in Yuelu District, Changsha China. The average ammonia con­
centrations were 40 and 75 mg/m3 for WTS and PT, respectively. The
WTS and PT had a space size of about 25 m3. The average temperature
was 32 ℃. There were five treatments including B1 pure culture, J1 pure
culture, B1 and J1 co-culture, water treated and untreated control (CK).
As the ammonia removing efficiency didn’t show a significant difference
between water and sterilized culture medium at WTS (Table S3),
therefore, we only used water to compare with different agents. When
used, 5 L of diluted agents (or water) were sprayed evenly into the air
using sprinklers, with a spray rate of 100 ml/s. The ammonia removing
experiments had tree independent replicates, with doing the experiment
for three days at the same time of the day (start at 11:00 a.m.). The air
ammonia concentration was analyzed using a portable gas analyzer
(measurement error: 3%) (Eranntex MS400, China). The ammonia
Y. Zhang et al.
concentration was recorded every 10 min for 90 min. The ammonia
removing efficiency (ARE) was calculated according to the following
equation.
ARE (%) = (C0 - Ct)/C0 × 100%
Where, the ARE is the ammonia removing efficiency, the C0 is the
ammonia concentration before treatment, the Ct is the ammonia con­
centration after t minutes treated with agents.
2.5. Genomic analysis of ARMs
2.5.1. Genomic sequencing
The genomic DNA was sent to Guangdong Magigene Biotechnology
Co., Ltd. (Guangzhou, China) for whole genome sequencing. The
genomic sequencing was performed on the Illumina Novaseq 6000
platform and the Pacific Biosciences RSII sequencer (PacBio, Menlo
Park, USA). For Illumina Novaseq sequencing, sequencing libraries were
generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina®
(New England Biolabs, USA) following manufacturer’s recommenda­
tions and index codes were added. The library quality was assessed on
the Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY) and
Agilent 4200 (Agilent, Santa Clara, CA) system. At last, the library was
sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end
reads were generated. For PacBio Sequel II sequencing, qualified genomic
DNA was fragmented with G-tubes (Covaris) and end-repaired to pre­
pare SMRTbell DNA template libraries (with fragment size of >10 kb
selected by bluepippin system) according to the manufacturer’s speci­
fication (PacBio, Menlo Park, USA). Library quality was detected by
Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY) and
average fragment size was estimated on an Agilent 4200 (Agilent, Santa
Clara, CA). SMRT sequencing was performed on the Pacific Biosciences
RSII sequencer (PacBio, Menlo Park, USA) according to standard pro­
tocols. We obtained a total of 1,747,431,104 bp and 5,751,166,383 bp of
raw data for bacterial and fungal sample, respectively. The low quality
reads were removed by the SMRT2.3.0 (Chin et al., 2013), resulting in 1,
400,668,930 bp and 4,817,706,638 bp high quality data, for bacterial
and fungal sample, respectively.
2.5.2. Genome assembly
The high-quality reads obtained from PacBio sequencer were
assembled into contigs using Canu 2.0 (https://github.com
/marbl/canu) (Koren et al., 2017) with default parameters; then the
contigs, together with the high-quality reads obtained from
second-generation sequencing were assembled into scaffolds and the
sequencing errors were correct using NextPolish (https://github.com/N
extomics/NextPolish) (Hu et al., 2019) with default parameters.
2.5.3. ORF prediction
For bacteria, the GeneMarkS (http://topaz.gatech.edu/GeneMark/
genemarks.cgi) (Besemer et al., 2001) was used to retrieve the related
coding genes. For yeast, the metagene (Noguchi et al., 2006) (http
://weizhongli-lab.org/metagenomic-analysis/server/metagene/) pro­
gram was used to retrieve the related coding genes.
2.5.4. ANI identification
The bacterial strain was identified according to Average Nucleotide
Identity (ANI) based on genomic sequences. The ANI analyses were
performed with the software JSpecies (Richter and Rossello-Mora,
2009). ANI was calculated using the MUMmer algorithm implementa­
tion, version 3.20 (i.e., ANIm) (Delcher et al., 2002).
2.5.5. Function annotation
We used three databases to annotate the coding gene functions. They
were KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Cluster
of Orthologous Groups) and KOG (Eukaryotic Orthologous Groups).
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Ecotoxicology and Environmental Safety 218 (2021) 112287
KEGG annotation was performed with Automatic Annotation Server Ver.
2.1 (https://www.genome.jp/kaas-bin/kaas_main) using bi-directional
best hit method. The COG and KOG annotation were performed on
WebMGA services platform, using an e value of 0.001.
3. Results and discussion
Ammonia is an air pollutant and odorous. Traditionally, air ammonia
treatment is based on chemical and/or physical processes, both are
expensive and produce secondary pollutants. Bioremediation of polluted
environments has gained more and more interest, so does the controlling
of the odorous gas. It has been demonstrated that using biomaterials as
packing medium in a biofilter was an efficient approach for filtering
ammonia (Zhou et al., 2012; Krivolapov et al., 2019). Some researchers
have used microbial strains and enzymes to reduce odor emission (Zhao
et al., 2017; Sun et al., 2020). It is also demonstrated that microbial
extracts, such as yeast-derived extract can remediate odorous gases (Kim
et al., 2020). Different to these approaches, we used microbial flora as
agents to remove air ammonia at two low ventilation places that suf­
fered from high ammonia (~100 ppmv) contamination. Our results
demonstrated that the Lactobacillus paracasei B1 and Pichia sp. J1
showed synergy in ammonia removing and the microbial flora agents
are effective solutions for treating ammonia pollution on site. Genomic
analysis indicated that the nutrients complementary and signal trans­
duction may regulated the synergy behavior between B1 and J1.
3.1. Isolation, selection and identification of ammonia removing
microorganisms
The ammonia removing microorganisms (ARMs) were isolated from
the waste leachate. We isolated 16 bacterial and 6 fungal strains from
the waste leachate. The ammonia removing experiment in the labora­
tory (Supplementary file) showed that the bacterial strain B1 and fungal
strain J1 had the highest ammonia removing efficiency (Table S3). The
B1 and J1 strains were used to make the microbial flora agents, resulting
in three agents, namely B1-pure culture, J1 pure culture and J1-B1
coculture.
Phylogenic analysis of 16S rDNA (Fig. S2) indicated the bacterial
strain B1 had the closest phylogenic relationship with genus Lactoba­
cillus, indicating it is a typical lactic acid bacterium.
The phylogenetic tree of 18S rDNA sequences (Fig. S3) showed the
fungal strain J1 belonged to the genus Pichia, a yeast fungus. Average
nucleotide identity (ANI) analysis has recently been proposed as a new
standard for inferring robust taxonomic relationships between bacterial
species based on genome comparison (Konstantinidis and Tiedje, 2005)
and it has been assumed that values of 95% or 95–96% for ANI corre­
spond to the 70% of the DNA-DNA hybridization reassociation value for
demarcating bacterial species. ANI analysis of the B1 strain genome
showed that the Lactobacillus sp. B1 has the highest similarity (ANI >
96%, Table S4) with Lactobacillus paracasei, therefore, the B1 strain was
named as Lactobacillus paracasei B1. The genus Lactobacillus is members
of the family Lactobacillaceae, and are psychrophilic, non-spore form­
ing, rod shaped, non-motile, Gram positive and facultative anaerobic.
Lactobacillus spp., the lactic acid bacteria, are important ingredients in
many food products and can ferment carbohydrates into lactic acids and
do not produce CO2. Acid–base neutralization is the main stairgate for
removing atmospheric ammonia, therefore, the Lactobacillus sp. B1 had
great application potential in ammonia purification. As the ANI could
not be used to identify fungus, we didn’t use it to classify the J1 strain,
but the Pichia sp. J1 is a typical yeast fungus. It is reported that yeast
increased the cell numbers of lactic acid bacteria during milk fermen­
tation (Álvarez-Martín et al., 2008), which meant that yeast was able to
promote the growth of lactic acid bacteria. Thus, we designed a yeast
and LAB (J1 and B1) co-culture system which was proposed to have
better ammonia purification capacity than pure cultures.
Y. Zhang et al.
3.2. Microorganisms are ideal materials for air ammonia removing
To prepare the biological flora agents, L. paracasei B1 and Pichia sp.
J1 was fermented in pure and co-culture systems, respectively. The
fermentation agents were applied at two public places, the waste
transfer station and public toilet to removing the air ammonia, and the
results are shown in Fig. 1. The growth curves of L. paracasei B1 and
Pichia J1, and pH value in pure and co-culture fermentation systems
during the 3-day fermentation period, are shown in Fig. 2.
Three agents including two (B1 and J1) pure culture agents and one
co-culture agent, reduced the air NH3 concentration significantly
immediately after the application of agents, then the air NH3 concen­
tration increased. Among all the treatments, the co-culture agent had the
best effect in reducing air NH3 and could reduce air NH3 for the longest
time (about 90 min), indicating the B1 and J1 were functionally coop­
erated to remove air ammonia. Compared to the control and water
treatment, the two pure cultures could significantly reduce the air
ammonia concentration, and their effects in reducing NH3 could last for
40–50 min. The two pure culture agents had similar ammonia removing
efficiency (ARE) most of the time (i.e. 0, 10, 40 and 50 min), whereas, at
20 and 30 min the B1 agent had significantly higher ARE than the J1
agent. The co-culture agent had a maximum ARE of 98.78% at the very
beginning, which was significantly higher than that of B1 (maximum
ARE of 78.93%) and J1 (maximum ARE of 75.00%) pure culture. The
maximum ARE of the co-culture agent was also higher than that of the
biofilter (92%) (Krivolapov et al., 2019), the wet scrubbers (84.3%)
Fig. 1. Ammonia concentration change (a and c) following the application of different agents and the ammonia removing efficiency (b and d) at waste transfer
station and public toilets. Time 0 was the time point immediately after application of bioagents. Results are means and S.D of three replicates. Different letters above
the column indicate the differences are significant at p < 0.05 level.
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Ecotoxicology and Environmental Safety 218 (2021) 112287
(Ashtari et al., 2016) and the environmental improvement agents (yeast
derived extract, 53.4%) (Kim et al., 2020). The results indicated that
spraying microbial agents is an ideal approach for removing odorous
ammonia from the air in public places.
Ammonia is relatively water soluble and pure water has the ability to
adsorb about 20% of the air ammonia (Ocfemia et al., 2005). The results
also showed that the water could reduce air ammonia of 15–20 kg/m3
(Fig. 1), and had a maximum ARE of 23.20% and 33.34% immediately
after application. However, the ARE is highly pH dependent, and an
acidic solution can enhance the ammonia absorption efficiency (Ashtari
et al., 2016). The excess ammonia water could recycle into liquid fer­
tilizers. The ARE was closely related to solution pH values, the lower the
pH of the solution the higher the ARE is (Fig. 2). That could be the reason
that the B1 pure culture had significantly higher ARE than the J1,
because the pH in B1 pure culture was significant than that in J1 pure
culture. Moreover, the pH in the co-culture system that had the lowest
pH values possess the highest ARE compared to other agents. Consid­
ering that the fungal strain produced little acids whereas the B1 is a
lactic acid producing bacteria, the lower pH in co-culture agents must be
a result of high bacterial B1 cell density (Fig. 2), but the high cell density
of B1 in co-culture system was because of the inoculation of J1, therefore
the fungal J1 contributed to the lower pH in co-culture agents by pro­
moting the growth of B1 bacteria. Hadlocon et al. (2014) used an acidic
solution with pH 1.48 to remove ammonia, and the maximum ammonia
removal efficiency can reach up to 86%, which is, however, much lower
than in our study (e.g. 98.78% for co-culture agents). Considering the
Y. Zhang et al.
Fig. 2. Growth curves of Lactobacillus paracasei B1 and Pichia sp. J1 (a), pH values (b) and dissolved oxygen (c) in pure and co-culture fermentation systems, and
microscopic observation of microorganisms (d). Results are mean and S.D of three replicates. In the co-culture fermentation system, the J1 strain was inoculated after
12 h cultivation of B1.
co-culture agents had higher pH (3.0), one could expect that there are
some other mechanisms involving in ammonia removal, such as cell
adsorption or microbial assimilation. In addition, concerns were
expressed about high acidity scrubbing solutions due to sulfuric acid
cost, safety, corrosion and environmental problems. Using microor­
ganisms to produce organic acids is safe, chip and environment friendly.
In our study, acids are the metabolic products of microbes (e.g. lactic
acid bacteria), therefore, one could not neglect the role of microbes. In
other word, the higher ammonia removing efficiency of agents is the
result of metabolic behavior (organic acid producing) by
microorganisms.
The results showed that compare to the pure cultures (3.57 × 108
cfu/ml for L. paracasei B1 and 8.17 × 108 cfu/ml for Pichia sp. J1,
respectively), the maximum cell density of L. paracasei B1 and Pichia sp.
J1 in co-culture system was significantly higher (10.67 × 108 and
11.00 × 108 cfu/ml, respectively), indicating the two strains promoted
the growth of each other. In the co-culture agents, the adhesion of LAB to
yeast was clearly observed (Fig. 2d). In addition, the stationary phase of
both strains was delayed in the co-culture system. The results in
consistent with other findings in food fermentation systems or food
waste treatment, that the yeast fungi were often used to promote the
growth of functional bacteria (e.g. lactic acid producing bacteria)
(Mendes et al., 2013; Sundberg et al., 2013).
Even though the acid produces one of the main approaches for mi­
crobes to remove air ammonia, the bacterial could also assimilate
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Ecotoxicology and Environmental Safety 218 (2021) 112287
ammonia to form glutamine and oxidize ammonia to form nitrate. The
mutual enhancement behavior between B1 and J1 in co-culture agents
could be the cooperation in many aspects (Deveau et al., 2018). How­
ever, the issues remained unclear.
3.3. Ammonia removal mechanisms of yeast J1 and bacteria B1
To reveal the ammonia removal mechanisms and the cooperative
mechanism of L. paracasei B1 and Pichia sp. J1, we sequenced the whole
genome of two strains using PacBio SMRT and Hiseq sequencing tech­
nology. The genome size for L. paracasei B1 and Pichia sp. J1 was
3,160,811 bp and 11,622,210 bp and GC content was 46.35% and
30.6%, respectively (Fig. 3). A total of 3, 133 and 6868 genes (CDs) were
predicted for L. paracasei B1 and Pichia sp. J1, respectively. The function
of the strains was investigated by annotating the CDs with KEGG and
COG database (Fig. S4). The results showed that 47.46% of B1 genes and
45.91% of J1 genes could be annotated in KEGG database, and 77.82%
of B1 genes and 46.75% of J1 genes could be annotated in COG
database.
The functional genes related to ammonia removal were shown in
Fig. 4. Neither L. paracasei B1 nor Pichia sp. J1 had ammonia oxidation
ability, but they could assimilate ammonia to form glutamine, which
suggested that other than water absorption and acid neutralization,
ammonia assimilation was also the main mechanism for microbial
agents to remove air ammonia. The results indicated that the J1 agent
Y. Zhang et al. Ecotoxicology and Environmental Safety 218 (2021) 112287

Fig. 3. Genomic features of Lactobacillus paracasei B1 and Pichia sp. J1.

Fig. 4. Genes involving in ammonia removing in Lactobacillus paracasei B1 and Pichia sp. J1.

had much higher ARE than the pure water because it could assimilate
the ammonia, but compared to the L. paracasei B1, it did not produce
acid components which led to lower ARE of J1 culture than the B1
culture. The ammonia removing efficiency of pure culture agents was
lower than the co-culture agent, suggesting that there were some
collaboration mechanisms between lactic acid bacteria (LAB) and fungal
yeast. This could be due to either signal transduction, complementary of
nutrients or adhesion proteins. Our results suggested that the acid pro­
ducing ability of LAB was enhanced by yeast fungi, in another word, the
high ammonia removing ability of co-culture agents was caused by the
synergy behaviors between yeast and LAB. Moreover, the ammonia
removing efficiency of pure culture agents went done sharply in
30–40 min, but last at a relatively high level for co-culture agents
(Fig. 1b&d) after 80 min of treatment. The results suggested that there

6
Y. Zhang et al.
were some other effects or than the acid-base neutralization, which
could be adsorption, absorption or assimilation.
3.3.1. TCA cycle and organic acids production
The TCA cycle was uncompleted in L. paracasei B1 but was complete
in Pichia sp. J1. The L. paracasei B1 lacked the malate to oxaloacetate,
the oxaloacetate to citrate, the citrate to oxoglutarate, and the oxoglu­
tarate to succinate pathways. Uncomplete TCA cycle in L. paracasei B1
led to accumulation of succinate and fumarate. Also, the L. paracasei B1
is a typical acetate production bacterium, possesses the complete acetate
production pathway which metabolize the acetyl-CoA to acetyl-P (pta
gene), and subsequently to acetate (acyP gene). Therefore, the acidifi­
cation in B1 pure culture system was the result of acetate accumulation.
The Pichia sp. J1 is a typical yeast, which has the complete TCA cycle and
the final production of fatty acid degradation is ethanal.
3.3.2. Ammonia assimilation
Both L. paracasei B1 or Pichia sp. J1 has the ammonia transporter,
amtB gene. The L. paracasei B1 has the glnA gene, which assimilates the
ammonia with L-glutamate to form L-glutamine. The L-glutamine is
deaminated by gltB and gltD genes to form L-glutamate that could
assimilate the ammonia. The Pichia sp. J1 has the same ammonia
assimilation function as L. paracasei B1, but the deamination of L-
glutamine is regulated by glt1 gene. The other difference between the
two strains is the Pichia sp. J1 can use L-glutamine for amino acid syn­
thesis, whereas the L. paracasei B1 cannot.
3.4. The interaction between B1 and J1
The co-culture of B1 and J1 in the fermentation system and the
ammonia removing experiment both showed cooperative behaviors (i.e.
promoting the growth in fermentation system, and enhancing the
Fig. 5. Signal transduction between Lactobacillus paracasei B1 and Pichia sp. J1, as predicted by genomic analysis.
7
Ecotoxicology and Environmental Safety 218 (2021) 112287
ammonia remove ability in site application experiment, of each other)
between bacterial B1 and yeast J1. Also, the inoculation of fungal J1
upregulated the acid producing ability of B1. The yeast fungi are widely
used in dairy, food, and beverage fermentations to promote the growth
of lactate acid producing bacteria (Maligoy et al., 2008; Sieuwerts et al.,
2010). It is proposed that the interaction (cooperative) mechanisms
included many aspects (Mendes et al., 2013), such as complementary
carbon source, acidic environment establishment, and amino acid ex­
change. Our results indicated that there might be carbon source ex­
change between B1 and J1. Although both B1 and J1 could use glucose
as carbon source (glycolysis, Fig. 3), the B1 did not have a complete TCA
cycle, therefore the intermediate products of TCA cycle, such as malate
and succinate, in J1 can be excreted and be utilized by B1 to produce
L-lactate. This could be the main reason that when co-cultured with J1,
the growth of B1 was significantly promoted. Accumulation of lactate
acid led to lower pH in co-culture system, and therefore, higher
ammonia removing efficiency of co-culture agents, compared to pure B1
culture.
Other than cooperated in using carbon sources, the results also
suggested possible interactions of L. paracasei B1 and yeast Pichia sp. J1
at nitrogen metabolism, particularly amino acid metabolism. The B1 did
have valine, leucine and isoleucine metabolic pathways, whereas the J1
have could synthetic valine, leucine and isoleucine using pyruvate, the
intermediate product of TCA cycle (Fig. 4). The results indicated that the
B1 and J1 cooperated in amino acid biosynthesis and utilization. This is
inconsistent with the finding in Lactobacillus and Saccharomyces, that the
Lactate acid bacterium. Lactobacillus could take up the amino acid
excreted by the yeast Saccharomyces, and suggested the interactions in
amino acid metabolism between LAB and yeast (Maligoy et al., 2008).
Signal transduction, such as quorum sensing, was an important
approach to the cooperative mechanism between microorganisms
(Papenfort and Bassler, 2016), which was seldom studied in the
Y. Zhang et al.
yeast-LAB co-culture system. Both B1 and J1 possessed an autoinducer 2
(AI-2) quorum sensing system (Fig. 5) for signal transduction. The LuxS
(S-ribosylhomocysteine lyase) genes that encode AI-2 signals, were
highly conserved in the B1 and J1 genome, which indicated that there
were AI-2 regulated signal transduction between L. paracasei B1 and
yeast Pichia sp. J1. The signal transduction, therefore, mediated the
cooperative behaviors (Rendueles and Ghigo, 2012) between L. para­
casei B1 and yeast Pichia sp. J1. One thing remains unclear was the ac­
ceptors of LuxS were not clear in neither L. paracasei B1 nor yeast Pichia
sp. J1, but it is highly possible that the LuxS may play roles in regulating
the carbon source and amino acid exchange between L. paracasei B1 and
yeast Pichia sp. J1. Furthermore, the auto-inducer peptide (AIP, bacte­
riocin) produced by L. paracasei B1 could be recognized by the Agr
acceptor system in yeast Pichia sp. J1. The AIP signal then regulated the
protein synthesis (e.g. EPS or adhesion proteins) in yeast. The EPS and
adhesion proteins affect the physiological and biochemical character­
istics of the LAB. The LAB cells could adhere to the yeast cell wall, as
observed in the co-culture agents (Fig. 1d).
4. Conclusion
To summary, in the present study, we obtained a yeast strain, Pichia
sp. J1, and a LAB strain, Lactobacillus paracasei B1, which are ideal mi­
croorganisms for atmospheric ammonia removal. moreover, the LAB
and yeast coculture agent has better effects than the pure culture(s). The
cell density of both B1 and J1 in the co-culture system was significantly
higher than that in the pure cultures, indicating they were synergic.
When cocultured with the yeast, the acid production ability of LAB was
also promoted. Therefore, the better ammonia removing ability of the
coculture agent was the result of synergic behavior between B1 and J1.
Genomic analysis indicates that the two strain could cooperate in carbon
and nitrogen sources utilization. It is also proposed that the quorum
sensing system also plays a role in regulating the synergy.
Suggested reviewers
Zhili He zhili.he@ou.edu, Qiang He Qianghe@utk.edu, Ye Deng
yedeng@rcees.ac.cn, Qingyun Yan yanqy6@mail.sysu.edu.cn, Baolan
Hu blhu@zju.edu.cn.
Funding
This work was supported by the Biodiversity Survey and Assessment
Project of the Ministry of Ecology and Environment, China (Grant
number: 19HJ2096001006), the National Natural Science Foundation of
China (Grant number: 41807332 and 41877345), the Hunan Interna­
tional Scientific and Technological Cooperation Base of Environmental
Microbiome and Application (Grant/Award Number: 2018WK4019)
and the Innovation and Entrepreneurship Center at CSU (Grant number:
502390003).
CRediT authorship contribution statement
Yanfang Zhang: Writing - original draft, Data curation, Investiga­
tion, Formal analysis. Zhimin Dai: Resources, Investigation. Huaqun
Yin: Conceptualization, Supervision, Funding acquisition. Min Zhang:
Methodology, Investigation. Hetian Zhang: Methodology, Investiga­
tion. Zhicheng Zhou: Supervision, Resources. Yongjun Liu: Resources,
Formal analysis. Qian Li: Conceptualization, Validation. Xiaolong Nan:
Conceptualization. Xueduan Liu: Conceptualization, Supervision.
Delong Meng: Conceptualization, Writing - original draft, Project
administration, Writing - review & editing, Software.
Declaration of Competing Interest
The authors declare that they have no known competing financial
8
Ecotoxicology and Environmental Safety 218 (2021) 112287
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We would like to thank The Hunan International Scientific and
Technological Cooperation Base of Environmental Microbiome and
Application (Grant/Award Number: 2018WK4019) and the Innovation
and Entrepreneurship Center at CSU (Grant number: 502390003) for
funding the project.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2021.112287.
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