A new Desmodesmus sp. from the Tibetan Yamdrok Lake.

Revegetation of exposed sub-soil, while a desirable recovery strategy, often fails due to extreme soil chemical properties, such as low organic matter and pH levels. Microalgae play a key role in maintaining water quality in the lakes and rivers on the Qinghai-Tibet plateau. Plateau microalgae have extensive application prospects in environmental purification, biotechnology, medicine and cosmetics, food industry, and renewable energy. To identify the high biomass of microalgae present in nature, microalgae with the greatest biomass were screened from natural water samples through filtration, pre-culture, and plate scribing separation. Following identification via 18S rRNA sequencing as for the Desmodesmus sp., we constructed a neighbor-joining phylogenetic tree. The novel Desmodesmus sp. from the Tibetan Yamdrok Lake were identified through polyphasic taxonomy. Simultaneously, the sequence of the experimental samples and the target species were shown different following the identification and analysis of SNP and InDel loci. The light-absorbing properties of plateau Desmodesmus sp. have been investigated previously. The characteristic absorption peak of Desmodesmus sp. on the plateau was measured at 689 nm in the visible spectrum using full wavelength scanning with a UV-Vis spectrophotometer. For Desmodesmus sp. which is prone to settling in the process of amplification culture. By monitoring the change trend of total nitrogen, total phosphorus, pH and electrical conductivity in algae solution system, we determined that the logarithmic growth phase and the best transfer window of Desmodesmus sp. were at 15-20 days. This study can provide basic research methods for the study of microalgae in high altitude areas, and lay a foundation for the later study and application of microalgae.

Introduction

The Qinghai-Tibet Plateau is the highest plateau worldwide, with an average altitude of over 4000 m. It is characterized by a unique physical and geographical environment with cold temperatures, hypoxia, little precipitation, long sunshine, and strong solar radiation [1]. Rivers and lakes are distributed at different elevation gradients, and microalgae on the plateau play a key role in maintaining the water quality of these lakes and rivers [2].

Recently, wastewater treatment based on microalgae has attracted increasing attention due to its environmental friendliness and potential economic benefits [3]. Studies have shown that microalgae can remove various pollutant from wastewater, such as oxygen consuming pollutants, nitrogen, phosphorus, heavy metals, organic matter, and absorb harmful gases such as NOX, SOX, and H2S at a certain concentration. The biomass harvested from wastewater treatment can also be utilized as raw materials for biofuel, feed, and chemical compounds [4-9]. Microalgae can use CO2 during photosynthesis as the only carbon source for heterotrophic growth and can use external carbon sources for heterotrophic growth to improve the biomass and oil content of microalgae [10, 11], which can greatly reduce global emissions of greenhouse gases such as CO2 [12].

Desmodesmus sp. was collected from Yamdrok Lake, Langkazi County, Shannan City, Tibet Autonomous Region, named YH-1 and subsequently purified, amplified, and cultured. This area belongs to the semi-arid climate area of the plateau sub-cold zone, with lower annual average temperature, lower air density, lower oxygen content and strong solar radiation. The growth rate and cell components of vegetation and microalgae growing in this area are vastly different from those of crops in plain regions [13].

A microalgae from Yamdrok Lake with the highest biomass was screened through filtration, pre-culture, and the plate separation method. It was identified as Desmodesmus sp. using 18S rRNA sequencing. Genotyping was determined through PCR amplification and locus mutation (based on GATK analysis). The characteristic absorption peak was determined by studying the light absorption characteristics of Desmodesmus sp. on plateau. The linear relationship between the concentration of Desmodesmus sp., the absorbance as well as the turbidity were established using the optical density method, and a regression equation was established. By analyzing total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD), pH, and electrical conductivity (EC) in the solution system during the course of amplification culture of Desmodesmus sp., the logarithmic growth end and the optimum transfer time of Desmodesmus sp. were determined.

Our results aid in understanding the isolation, purification, amplification, and culture methods of microalgae in high altitude areas. For some microalgae species that settle easily and affect the accuracy of absorbance value (such as Desmodesmus sp.) [14], the best transfer species or harvest time can be accurately determined by monitoring the TN, TP, COD, EC, and pH indicator values during the culture process. Using the linear relationship between microalgae concentration and absorbance as well as turbidity, the microalgae concentration can be determined, which can provide the basis for the follow-up large-scale production and application research. Using polyphasic taxonomy to identify Desmodesmus sp. from the Tibetan Yamdrok Lake. Through the identification and analysis of SNP and InDel sites to find the differences from that of the target species.

Materials and methods

Collection of microalgae

One litre of water was collected from the Yamdrok Lake (25 times by water collector), the phytoplankton were filtered through a phytoplankton net of 500 mesh, and the water sample was poured into a Teflon bucket of 50 L for preservation. Then, 200 g of sediment from the bottom of the lake was randomly collected at various points, and stored in a Teflon bottle with a breathable film tied at the mouth. Transport to the laboratory was completed within 6 h.

Preparation method of culture medium

To prepare the liquid culture medium, we added 1.7 g BG 11 culture medium (Haibo Bio, Qingdao, China) into a 1000 mL conical flask, added distilled water to a constant volume, shook until the culture medium powder was completely dissolved. A 1 M HCl or NaOH solution was used to adjust the pH to 7.2. Then, we covered the bottle mouth of the conical flask with a rubber stopper, and then used a 0.22 μm sterilization membrane for filtration on a clean workbench, following which it was stored for later use.

To prepare the solid culture medium, we added 25 g of nutrient agar medium (Haibo Bio, Qingdao, China) to the liquid culture medium, heated it to 60°C in water bath, and shook it until it was completely dissolved. Next, we used a 0.22 μm sterilization filter membrane for filtration on a clean workbench, following which it was stored it for later use. After the culture medium cooled to 35°C, it was poured into a sterile petri dish and cooled continuously to obtain a solid culture medium.

Pre-culture after filtration

Under open purification workbench conditions, the collected water samples were poured into the Brinell funnel and filtered under the suction filtration of the water ring vacuum pump using a 0.45 μm water-based filter membrane.

Each 100 L water sample was filtered thrice, with a new filter membrane being used each time. The replaced filter membrane was placed into a liquid culture medium prepared in advance for culture. Three groups of parallel cultures can be obtained using the three filtration steps. There was no need to filter the bottom mud collected from the water bucket; approximately 30 g of bottom mud was taken from each group and incubated into liquid culture medium separately, producing another three parallel groups. The top of the conical flask was sealed with a breathable sealing film to prevent external bacteria from entering.

The incubated samples were shaken thhrice a day in a light incubator with a temperature of 25 ± 1°C (light-dark ratio of 12 h:12 h, light intensity of 2000 Lx), and each sample was randomly placed to receive light evenly. After culturing for 7–15 days, until the entire solution system was yellow-green or green, microscopic examination and dilution were carried out to separate microalgae strains [15].

Isolation and purification of microalgae

When the purification workbench was turned on, the aforementioned fresh algal liquid at the end of logarithmic growth was dipped for approximately 10 days with the inoculation ring, and a dense line was drawn on the solid medium. Samples were inverted in a 25 ± 1°C light incubator for culture (light dark ratio 12 h:12 h, light intensity 2000 Lx). The formation of microalgae strains with large biomass was observed after 7–10 days. Large algae colonies were collected for microscopic examination. Microscopic identification of algae indicated that the separation was successful [16, 17].

① In a 1,000 mL beaker, 500 mL BG 11 liquid medium was prepared according to the formula for BG 11 medium, and the pH was adjusted to 7.1. Next, 200 mL of BG 11 liquid medium was filled in each 500 mL conical flask, or 50 mL of BG 11 liquid medium filled in 150 mL conical flask.

② During step ①, the BG 11 culture medium should be filtered through a 0.22 μm sterilization filter membrane on a clean workbench, and then the bottle mouth should be bandaged for later use. Gas permeable membrane, sterile filter membrane, filter device, conical flask, and the inoculation ring should be sterilized by steam pressure sterilization pot at 101.33 kPa and 121°C for 30 min before use.

③ On a clean workbench, the microalgae with good growth were selected using a sterile inoculation ring and incubated into 10 mL BG 11 liquid culture medium after sterilization and filtration at 25 ± 1°C and a light intensity of 2,000 Lx (light ratio is 12 h:12 h). Next, the microalgae sample in medium was shook or stirred 2–3 times every 12 h and cultured for 7–15 days. When the biomass was obviously increased (>106/L), amplification culture can be carried out.

④ On a clean workbench, 10 mL of cultured algae liquid was added into the liquid medium containing 20 mL BG 11 for culture (1:2 amplification) at a temperature of 25 ± 1°C and at 2,000 Lx (the light ratio is 12 h:12 h). Microalgae were cultured for 7–15 days by shaking or stirring 2–3 times every 12 h (bottle mouth was sealed with a gas permeable membrane and the biomass was obviously increased).

The scale-up cultivation of Desmodesmus sp.

Normal culture conditions: temperature 25 ± 1°C, light intensity 3,000 Lx (light ratio 12 h:12 h), shaking or stirring 2–3 times every 12 h. When the culture volume is greater than 1 L, consider adding stirring to assist the culture (culture time 7–15 days). When microalgae species with long culture period are used, the culture period can be appropriately prolonged.

⑥ Next, the culture solution from ⑤ was added to 150 mL BG 11 medium for amplification culture (1:5 amplification; culture period 15–20 days). When microalgae species with long culture period are used, the culture period can be appropriately extended to approximately 30 days.

⑦ For volumes of amplification culture medium greater than 1 L, the amplification culture conditions were 25 ± 1°C, the light intensity was 3,000 Lx (light ratio is 12 h:12 h) and 100–200 r/min. When microalgae species with long culture period are used, the culture period can be appropriately extended to approximately 30 days.

⑧ After the logarithmic growth phase of green algae on the plateau, when the final optical density at 689 nm (OD689) ≥ 0.70 (the maximum absorption wavelength of each algae species can be obtained through full-wavelength scanning using a UV-spectrophotometer), the algae solution after amplification was centrifuged or filtered and then washed with BG 11 culture solution for 3 times for later use [18, 19].

Species identification of 18S rRNA

Using the extracted microalgae DNA as the template, primers 1143-510-2-F (AATTGACGGAAKGGCA) and reverse 1637-510-2-R (CGACGGGCGGTGTGTA) were designed using the primer premier 5.0 software. The 18S rRNA gene was amplified using PCR. A total of 25 μL PCR reaction system was used, containing DNA template 1 μL, primers 1 μL each, dNTP 1 μL, Taq Buffer (with MgCl2) 2.5 μL, Taq enzyme 0.25 μL, and ddH2O to 25 μL. The PCR reaction conditions were: pre denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s (0.5°C per cycle), extension at 72°C for 30 s, 10 cycles; Denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; Recover and extend at 72°C for 10 min and kept warm at 4°C. After the PCR products were detected, the sequences were blast compared using the GenBank database. Next, we constructed a neighbor-joining phylogenetic tree based on 18S rRNA sequences [16].

Determination of amplification culture cycle

Microalgae consume nutrients such as N, P, and heavy metals during their growth process [20, 21]. Considering the easy sedimentation characteristics of Desmodesmus sp., the logarithmic phase of the growth, transfer, and harvest time of this specie were determined by detecting the change trends of TN, TP, pH, and EC in the culture solution of Desmodesmus sp. during the amplification culture stage; thus, preventing potential errors caused by the OD method [22].

The water quality multi parameter tester (KN—MUL20, Kenuokeyi instrument, Beijing) and intelligent digestion instrument (KN—HEA12, Kenuokeyi instrument, Beijing) were used to determine TN and TP. The water samples used to determine TN and TP were filtered through a 0.22 μm filter in advance. EC and pH were measured using a multi parameter test pen (PCSTestr—35, USA).

Experiment on correlation between algae concentration and absorbance and turbidity

To determine algal concentration a blood cell counting plate and an optical microscope were used to count. The average value from three counts was computed and the average deviation was controlled within 10%. Before counting, the algal solution was vibrated on an oscillator to ensure the uniform dispersion of microalgae, and the initial algal concentration (PCS/L) was determined by taking the stock solution of Desmodesmus sp. culture solution as the test water sample.

Method for preparing water sample containing algae: Dilute 10 mL of the same volume of algal solution with ultrapure water successively in 20 mL, 40 mL, 80 mL, 100 mL, and 200 mL. Dilute it into a series of algal solutions with different concentrations at constant volume to allow cell counting and turbidity detection (there are three parallel samples). Dilute 10 mL of the same volume of algal solution with ultrapure water to 20 mL, 30 mL, 40 mL, 50 mL, and 100 mL respectively, and dilute it into a series of algal solutions with different concentrations at constant volume for cell counting and OD689 measurements (there are three parallel samples).

According to the above values, fit the absorbance algae concentration curve and establish the linear regression equation. The same was done for algal turbidity and the concentration curve [23, 24].

Morphology identification

Methods for preparing samples for scanning electron microscopy

Scanning electron microscopy (SEM) can be used to directly observe the three-dimensional structure of the specimen surface and reflect the morphological characteristics of various cell surfaces and fracture surfaces. The pre-treatment of SEM cell samples includes sampling, fixation, dehydration among others.

Material preparation

For bulk samples, the observation surface should be as flat as possible without affecting the purpose of the experiment and the size should be 2–3 mm thick and 5–6 mm long and wide.

Fixing of samples. Cultured cells were centrifuged at 800–1500 RPM for 8–15 min to enrich the precipitation. The precipitation was immersed in PBS (0.1M, without NaCI), the cells or tissues were rinsed several times, the supernatant was removed by centrifugation, and 2.5–3% glutaraldehyde precooled at 4°C was added. Next, tissues were fixed at 4°C for 4 h or overnight (depending on the need of tissue size, this step can be extended), then the fixative was removed and samples were dipped into PBS (0.1M, without NaCI) 3–5 times, 15 min each time.

Dehydration

Dehydration was performed with a gradient series of alcohol (30%, 50%, 70%, 80%, 90%, 95%, and 100%), 10–20 min at each time concentration (generally 15 min), then thoroughly dehydrate with 100% alcohol 1–2 times, and then incubated with isoamyl acetate (banana water) twice (20 min each time).

Sample drying and conductive treatment

We used the critical drying method. Sample conduction treatment was applied using the vacuum spraying method (spraying should be uniform) after the completion of scanning electron microscope observation [25]. (Model: TEM Hitachi SU8010 (Japan), Leica DM500 biomicroscope (Germany))

Preparation method of ultrathin section for transmission electron microscopy

Materials and fixation

Cell sample: Cell mass should be at least half the size of a mung bean. The cells/bacteria were collected by centrifugation, the culture medium was discarded, 2.5% glutaraldehyde was added, and the cell mass was dispersed and stored at 4°C. For adherent cells, the culture medium was first decanted, 2.5% glutaraldehyde was added, fixed at 4°C for 15 min, scraped with cells, collected by centrifugation (fixative was retained), and stored at 4°C. Samples were fixed for at least 4 h.

Osmic acid fixation

Cells or tissues fixed with glutaraldehyde were rinsed trice with 0.1 M phosphate buffer (pH 7.2) for 15 min each time, and then fixed with 1% hic acid •0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature (20°C; the fixing time was adjusted appropriately for different samples). Next, samples were rinse trice with 0.1 M phosphate buffer (pH 7.2) for 15 min each time.

Dehydration

The samples were dehydrated using 30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100% (twice) alcohol, 15–20 min at each concentration (the dehydration time was appropriately extended for samples with more water content and thick cell membrane).

Seepage. The used osmotic agent was acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), epoxy resin, 37°C in the temperature box, 8–12 h each time.

Embedding

The permeated samples were placed into capsules or embedded plates, the embedding agent epoxy resin was added, and the samples were polymerized at 60°C for 48 h. The section thickness was 80–100 nm.

Double staining

Uranium-lead double staining (2% uranyl acetate in saturated aqueous solution, lead citrate, stained for 15 min at room temperature) was performed. Sections were dried overnight at room temperature and imaged using electron microscopy [26, 27].

Model: TEM Hitachi 7800 (Japan)

Ultra-thin slicer: Leica (Germany) Model: EM UC7

Identification and analysis of SNP and InDel loci

GATK software was used to identify base mismatches between transcriptome data and the reference genome of streptozoa (Assembly MH624152.1); thus identifying potential SNPs and InDel sites. GATK recognition criteria were as follows: (1) No more than three single base mismatches occurring within 35 bp; (2) The quality value after sequence depth standardization being greater than 2.0. Each sample was screened according to the above conditions and reliable SNP/InDel sites were finally obtained [28]. SnpEff software was used to annotate the mutations according to the annotation information of the reference genome, and the distribution of the mutation sites on the genome structure was statistically analyzed [29]. According to the location of the mutation site on the reference genome and the gene location information on the reference genome, the region where the mutation occurred within the genome and the impact of the mutation (synonymous mutation or non-synonymous mutation) were predicted. SNP sites can be divided into Transition and Transversion according to the mode of base replacement [30].

Results

Species identification using SEM and STM

In our culture environment, the individual cells of alga were oval in shape and appeared as two-cell aggregates and single cells. The cell wall decoration of this alga was analyzed using SEM. The most prominent morphological features were uninterrupted rib-like decoration and warts without protrusions.

As shown in Fig 1A, the algal cells were arranged into two-cell aggregates. The two cells of the aggregate were of similar size, with a length and width in the range of 10.3–12.5 × 3.2–3.4 μm, and they had a relatively high aspect ratio (3.5 ± 0.6 on average; Fig 1A). The lateral wall of the cell had a spinous protrusion that extended longitudinally as a whole and was approximately the same length as the cell (Fig 1A, arrow). The surface of the cell wall is similar to that of peanut shell and was densely ribbed in meridian and zonal directions. These ribs were formed by the thickening of the inner cell wall layer.

Fig 1

Morphological graph based on SEM of Desmodesmus sp.

(YH-1). All cells studied retained chloroplasts, which contained thylakoids with an electron clear lumen (Fig 5C and 5D, arrows). In the chloroplast structure, the visible starch grains formed an incomplete sheath that was attached to the chloroplast envelope (Fig 5A and 5C). Simultaneously, a small number of plastid globules were observed in the microdomains of thylakoid blebs, which were structurally connected to the extracorporeal membrane of the thylakoid (Fig 5C and 5D).

Fig 5

Ultrastructure of Desmodesmus sp. cells.

(a) Overall view of the cell. (b–d) Regions of chloroplasts. Ch: Chloroplast; Cw: cell wall; N: nucleus; Ob: oil body; P: protein nucleus; Pg: plastid pellet; S: starch grain; T: cell nucleus.

The length and width of cells appearing as single cells ranged from 5.5–5.8 × 1.7–2.0 μm, with an average aspect ratio of 3.0 ± 0.2 (Fig 1B–1D). Among them, two types of single cells were observed, with thorns and without thorns. The cell wall also possessed a spinous process, extending longitudinally as a hole. The cell wall surface was mainly distributed with meridional rib-like ornamentation. Spiny single cells had chimney-like projections at the poles of the cell (Fig 1B, asterisk) and similar projections on the lateral wall of the cell. The outer wall of the projections appeared fairly smooth and vertical. The spiny single cell had a chimney-like protrusions only at one pole (Fig 1C and 1D, asterisk). There were clearly visible depressions on the cell wall of the spiny single cell, which are presumed to be gaps left by the thickening of the cell wall (Fig 1C and 1D, thin arrows).

Species identification by 18S rRNA

Using the new generation sequencing method, DNA was extracted from the samples using the test kit produced by Sangon Biotech (Shanghai). The amplified PCR products were detected and purified through PCR amplification, agarose electrophoresis detection, gel recovery and other steps. The PCR products were sequenced using the 3730-xl sequencer produced by ABI (USA).

After PCR amplification, 18S rRNA was obtained. The length of the PCR amplification product: site sequencing primer was approximately 150–300 bp and 80–150 bp away from the site, while the exon detection primer was approximately 150 bp upstream and downstream of the exon. The PCR product band of target gene sequencing was generally shorter than 1200 bp. The login number used to analyze the sequence homology (NCBI) was PRJNA810921 and we discovered that our sequence was most closely related to Desmodesmus sp., with an 18S rRNA sequence homology of 99.6%. Through BLASt online comparison, we discovered 10 species sequences of Desmodesmus sp. species with a high similarity to YH-1 species (96.0%;).

To further analyze the evolutionary relationship of Desmodesmus sp., the 18S rRNA gene sequences of related species were downloaded from the GenBank database through sequence alignment and referring to NCBI annotation information. Simultaneously, according to the highly conserved characteristics of 18S rDNA, 32 sequences with high similarity and published sequences of algae genome related species were downloaded, the neighbor-joining phylogenetic tree was constructed using the Chloroella 18S rRNA (GenBank accession number: KC790435.1) and Chlorophyta 18S rRNA (GenBank accession number: HQ900842.1) outgroup sequences. The YH-1 and the Desmodesmus sp. (GenBank accession number: AB917136.1) clustered into one branch, and the two appear closely related. The Desmodesmus sp. with GenBank accession number KF673371.1 and MK541739.1 clustered into one branch. The Desmodesmus sp. with GenBank accession number: OK641939.1, MW077168.1, X73995.1, MK541740.1, MG022724.1, MW471025.1, MZ570911.1, AB917128.1, MW136451.1, MH624152.1, AB917097.1, and HM633188.1 clustered into one branch. After the aforementioned two branches clustered into one branch, they combined with the Desmodesmus sp. (GenBank accession number AB917136.1) and clustered into one branch, and then gather with YH-1 to form a large branch (Fig 2A and 2B).

Fig 2

Neighbor-joining phylogenetic tree based on 18S rRNA sequences.

Identification and analysis of SNP and InDel loci

The Trimmomatic trimer was used to process the raw data obtained from sequencing in order to generate clean data. Data quality control statistics were as follows: Total Reads Count(#), 24615292; Total Bases Count (bp), 3597867603; Average Read Length (bp), 146.16; Q30 Bases Ratio (%), 92.96%; GC Bases Ratio (%), 60.57%. The most prominent types of variants in the entire sample were A-G, T-C, G-A, and C-T (Fig 3A).

Fig 3

(a) Type and number of individual variants in the entire sample. (b) SNP/InDel distribution in the entire gene mountain was calculated using a 50 bp window. (c) Distribution of individual variants across the entire gene in the entire sample.

Considering 50 bp as the window for calculation, the distribution map of SNP/InDel in the entire gene mountain was constructed. The map indicates that there are great differences in the whole sequence range; thus, suggesting that the detected sample sequence is different from the target species sequence (Fig 3B).

The outer circle represents the size of the entire gene, with 50 bp as a small window. The second circle represents the distribution of each variant on the whole gene and its RMS mapping quality (MQ). The larger MQ represents the variant accuracy. The third circle represents the sequencing depth of each variant and the last circle represents the mutation conversion type. SNP mutation between purines or pyrimidines, are represented by 1, while SNP mutation between purines and pyrimidines, are represented by 0. SNP mutation between purines and pyrimidines, are represented by– 1; Where A > T and T > A are represented as the same type in the figure (Fig 3C).

The TP monitoring data indicates that the best transfer time for Desmodesmus sp. is 15–20 days after amplification. After 20 days, microalgae undergo apoptosis, which leads to increased TP index in the solution. The main reason for the increase in TP is the release of organic matter and metal elements caused by the decomposition of microalgae cells. Therefore, for the duration of 20 days, it is necessary to replant or harvest again ().

Fig 4

(a) Trend diagram of TP during the microalgae amplification stage. (b) Trend diagram of TN during the microalgae amplification stage. (c) Variation trend of pH value during the microalgae amplification and culture stage. (d) Variation trend of EC during the microalgae amplification stage.

The TN monitoring data suggests that best transfer time for Desmodesmus sp. is 15–20 days after amplification. After 20 days, microalgae undergo apoptosis, which leads to increased TN index in the solution. The main reason for the increase in TN is the release of organic matter and metal elements caused by the decomposition of microalgae cells. Therefore, over 20 days, it is necessary to replant or harvest again ().

The pH value of microalgae fluctuates during the amplification period, mainly because the growth process of microalgae relies on CO2 consumption from the atmosphere. The consumption of CO2 in the process of dissolution and photosynthesis in water will induce the change in the pH value of water. We observed that between 15–23 days, microalgae reached a balance between CO2 adsorption and consumption during the growth process; thus, reaching a stable growth state. The pH monitoring data indicates that the best transfer time for Desmodesmus sp. is 15–20 days after amplification. After more than 20 days, it is necessary to replanted or harvested again ().

The EC value of microalgae fluctuates during the amplification culture process, mainly because the growth process of microalgae relies on the consumption of metal nutrients from the culture medium; thus, resulting in a decrease in EC. Simultaneously, the natural growth of microalgae leads to an increase in EC, especially after the logarithmic growth phase as cell wall breaking will lead to the release of metal elements and ultimately an increase in EC. Based on the EC values obtained in this study, the best transfer time for Desmodesmus sp. is 15–20 days after amplification. After more than 20 days it is necessary to replanted or harvested again ().

Study on light absorption characteristics

After full wavelength scanning using a UV-Vis spectrophotometer, the stock solution from the Desmodesmus sp. culture solution demonstrated no obvious absorption peak in the UV region, while an obvious absorption peak was observed at 689 nm, that is, its characteristic absorption peak. The supernatant (background liquid) obtained following centrifugation of microalgae liquid culture medium was analyzed similarly, and the absorption curve was relatively stable without an obvious absorption peak; therefore, the characteristic absorption wavelength was concluded to be 689 nm. After the characteristic absorption peak was obtained, the relationship between the concentration of Desmodesmus sp. and the absorbance of culture solution was investigated (S1 Fig).

Relationship between Desmodesmus sp. concentration and absorbance of culture solution

Our results indicate a good linear relationship between the concentration of Desmodesmus sp. in the culture solution and the absorbance within a certain concentration range (106–108/L) at the maximum absorption wavelength of 689 nm. The concentration of microalgae in the culture process can be accurately derived based on absorbance values (S2 Fig).

Relationship between Desmodesmus sp. concentration and turbidity of culture solution

Our results indicate a good linear relationship between the concentration of Desmodesmus sp. in the culture solution and the turbidity within a certain concentration range (106–108/L). The concentration of microalgae in the culture process can be accurately derived based on turbidity (S2 Fig).

Discussion

Using water samples collected from Yamdrok Lake (4400 meters above the sea level), following filtration, pre-culture, as well as plate separation methods of which, we obtained microalgae strains with the maximum biomass after separation and purification. Following amplification and culture, the Desmodesmus sp. were microscopically observed and one microalgae strain with a relatively high biomass was selected.

By first filtrating and then pre-culturing before the plate separation steps, one can benefit from using simple equipment, simple operation, and reduced workload. The separated samples are likely to be obtained from two or more cells. The isolated single cell species of microalgae might not be the target species; however, other species or new species may be isolated. The used method is especially suitable for the preliminary separation of water samples from natural water areas, without being limited by the species and size of microalgae. Here we showed that we successfully isolated Desmodesmus sp. for the first time from Yamdrok Lake, Tibet.

Using polyphasic taxonomy to identify Desmodesmus sp. from Tibetan Yamdrok Lake

The surface morphology of the cell wall and the presence of spines are important features that distinguish Scenedesmus sp. from Desmodesmus sp. Species belonging to the Scenedesmus family have no spines and have undecorated cell walls. However, two species types from the Desmodesmus family, namely spiny species and small spiny non-spiny species. The most prominent morphological features were uninterrupted rib-like decoration and warts without protrusions, which were different from the morphological features of other Streptomyces cells in the literature [31-35].

The cells investigated in this study retained their ultrastructure intact (Fig 5A) and the cellular ultrastructure shown in Fig 5 is typical of the observed microalgae [36-39]. By analyzing the sequence homology from NCBI, we observed that the isolated microalgae was closely related to Desmodesmus sp., with a 18S rRNA sequence homology of 99.6%. Following BLASt comparison, we discovered 10 sequences of Desmodesmus sp. with high similarities to the YH-1 species (96.0%), which form a separate branch on the neighbor-joining phylogenetic tree [15]. ThroughBased on the polyphasic taxonomic identification method, we can conclude that the Desmodesmus sp. identified in Yamdrok Lake is a new species.

The identification and analysis of SNP and InDel sites indicate that the sequence of the experimental samples is different from that of the target species

The distribution and mutation types of these SNPs/InDels are illustrated using graphs in Fig 3 and shows that our SNP/InDel sequencing was of high quality and that the sequence of the sample was different from that of the target sequence. Furthermore, the mutation quality of the experimental sample and the reliability were high. The mutation types of the experimental samples were distributed throughout the entire sequence, and there were numerous types of mutations with very high quality. The number of SNPs and InDels in the entire sequence, the intuitive number of mutations at each position, the high-quality evidence, and the refined mutation type show that our experimental samples are significantly different from the target sequence at genetic level, which indicates that our experimental samples represent a newly discovered species of Desmodesmus sp.

Study on the growth cycle of amplification culture of Desmodesmus sp.

Next, we showed that the maximum characteristic absorption peak of Desmodesmus sp. was 689 nm. Furthermore, we showed that the culture medium of Desmodesmus sp. had a good linear relationship with the absorbance and turbidity.

During the culture process, Desmodesmus sp. exhibit obvious sedimentation; thus, being associated with errors when determining the logarithmic growth end through simple optical density method. By measuring the change trend of TN, TP, pH, and EC in the water during the amplification culture of Desmodesmus sp., we accurately assessed the end of the logarithmic growth phase as well as the best time for species transferring and picking [40].

After a 1:5 amplification culture, the changes in TN, TP, pH, and EC levels were monitored in real time for 30 days. We concluded that the best transfer time was 15–20 days after the amplification period. After more than 20 days, it is necessary to replanted or pick the microalgae again. Our findings provide novel insights into the methods used to expand the culture of microalgae.

Conclusions

Novel species of Desmodesmus sp. from Yamdrok Lake, Tibet were identified using polyphasic taxonomy. Simultaneously, the S18 rRNA sequences of the experimental samples and the target species were shown to be different by identifying and analyzing the SNP and InDel loci.

By measuring the change trend of TN, TP, pH, and EC of Desmodesmus sp. during the scale-up cultivation, the end of logarithmic growth as well as the best time of seed transfer and harvest can be accurately assessed.

Desmodesmus sp. has its characteristic absorption peak at 689 nm. Within a certain concentration range, the algae concentration in the culture solution has a good linear relationship with absorbance and turbidity; therefore, absorbance or turbidity can be used to determine the algae concentration during culture.

During the experiment, the bubbles produced by the shaking process and the sedimentation of Desmodesmus sp. also have a certain influence. When measuring the absorbance and turbidity, it is necessary to shake the algal liquid evenly to reduce the detection error caused by sedimentation of microalgae. Through the basic research of Desmodesmus sp., we provided the groundwork for the study of other microalgae.

Genotyping results.

(XLSX)

Click here for additional data file.

(a) Full wavelength scanning spectrogram of Desmodesmus sp. (b) Full wavelength scanning spectrum of background solution.

(TIF)

Click here for additional data file.

(a) Relationship between microalgae concentration and absorbance of culture solution. (b) Relationship between microalgae concentration and turbidity of culture solution.

(TIF)

Click here for additional data file.

12 Jul 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Vandana Vinayak, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf  and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ.

4. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

6. Thank you for stating the following financial disclosure:

“This research was funded by Research on environmental risk management and control of industrial solid waste recycling process in low temperature, low pressure and anoxic environment, grant number 2019YFC190410304 witch was funded by Sub project of major R&D plan of the Ministry of science and technology. This research was also funded by The central government supports the phased achievement funding of local university projects (ZCKJZ [2021] No. 1, [2020] No.1, [2019] No. 44 and [2018] No.54).”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript titled "A new Desmodesmus sp. from Tibetan Yamdrok Lake" is interesting. However the following corrections are recommended:

1. How did you claim that the Desmodesmus sp. from Tibetan Yamdrok Lake is new? Validate the statement with concrete facts and findings.

2. Taxonomic keys are missing. Refer to the taxonomic credential with reference to the microalgae and the previous report.

3. The discussion needs to be strengthened in the light of the relevant literature available on the Desmodesmus sp.

4. The paper needs to corrected throughout to develop the flow and findings of the paper in the sequential pattern.

5. The paper requires major modification.

Reviewer #2: The authors describe their findings on a Desmodesmus species from Tibetan Yamdrok Lake in Tibet, China via a series of methods including strain isolation, molecular identification and determination of culture conditions. Totally speaking, the authors should clearly emphasize the novelty of this finding in the manuscript. Besides, the following comments are also for your references to improve the quality.

1. ‘sp.’ should not in italic form.

2. The resolution of figures is too low to provide information.

3. The data should be carried out in triplicate, and error bars should be provided.

4. In the M&M section, the general process of experimental methods should be concise by referring the published paper.

5. The initial value of TN in Figure 3c should be 200-260 ppm. Please check.

6. There is almost no discussion within the manuscript. The authors should emphasize why they perform this work. What is the significance?

7. The format of the references needs to be unified.

8. There are spelling, grammar and formatting errors in this article, and it should be applied to a professional editing service for language improvement.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Archana Tiwari

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Aug 2022

Dear Vandana Vinayak,

Thanks for providing us with this great opportunity to submit a revised version of our manuscript. We appreciate the detailed and constructive comments provided by the reviewers. We have carefully revised the manuscript by incorporating all the suggestions by the review panel.

We have read the reviewers’ and your comments carefully and have made revision which marked in red in the manuscript. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. Here, we would like to explain the changes briefly as follows: We have rewritten the article based on your comments, placing more emphasis on the methods, results, discussion and conclusion. In the remainder of this letter, we discuss each of your comments individually along with our corresponding responses. We have written a point-by-point response letter for two reviewers, you can see the details at the end of this letter. In all, we found these comments are quite helpful. And special thanks to you and reviewers for your good comments again.

Reply to Reviewer #1

Dear Reviewer,

Thank you very much for giving us an opportunity to revise our manuscript, and we also appreciate you very much for your positive and constructive comments and suggestions on our manuscript.

Comments: “The manuscript titled "A new Desmodesmus sp. from Tibetan Yamdrok Lake" is interesting. However the following corrections are recommended:

1. How did you claim that the Desmodesmus sp. from Tibetan Yamdrok Lake is new? Validate the statement with concrete facts and findings.

2. Taxonomic keys are missing. Refer to the taxonomic credential with reference to the microalgae and the previous report.

3. The discussion needs to be strengthened in the light of the relevant literature available on the Desmodesmus sp.

4. The paper needs to corrected throughout to develop the flow and findings of the paper in the sequential pattern.

5. The paper requires major modification.”

Thank you very much for your opinion. We have rewritten the article based on your comments, placing more emphasis on the methods, results, discussion and conclusion. In the remainder of this letter, we discuss each of your comments individually along with our corresponding responses. To facilitate this discussion, we first retype your comments in italic font and then present our responses to the comments.

Comment 1: How did you claim that the Desmodesmus sp. from Tibetan Yamdrok Lake is new? Validate the statement with concrete facts and findings.

Response 1: Thank you very much for your professional review, which was a great help in revising the article. The morphological identification of microalgae is the key and difficult point of the Desmodesmus sp.. We have highlighted this work in the article, and the method, results and discussion are presented in the article. Through the identification and analysis of SNP and InDel sites, it was further clarified that there were obvious SNP and InDel sites differences between theDesmodesmus sp. found in Yamdrok Lake and the existing algal strains of the same species, which proves its new characteristics in adapting to the special environment of high altitude., which from the side verified its new characteristics in adapting to the special environment of high altitude. Specifically, databases for identification were described in the “Result” and “Discussion”.

Comment 2: Taxonomic keys are missing. Refer to the taxonomic credential with reference to the microalgae and the previous report.

Response 2: Your comment reminds us to focus on the description of taxonomic credential, which is crucial to the structure of the article. In this modification, we added the experiments of STM and SEM and described the morphology of Desmodesmus sp. in detail. Combined with the polyphasic classification method, we conducted an in-depth analysis and discussion of microalgae. We have rewritten the Results section to describe the research results in detail. On the other hand, we also explain the obtained results in detail and with detailed explanations of its importance.

Comment 3: The discussion needs to be strengthened in the light of the relevant literature available on the Desmodesmus sp.

Response 3: Thank you for your valuable advice, which will make the revision of the article structure more specific. We rewrote the discussion section and cited important references to discuss the findings in detail.

Comment 4: The paper needs to corrected throughout to develop the flow and findings of the paper in the sequential pattern.

Response 4: Thank you for the detailed review. We carefully modified the methods, results, discussion and conclusion sections. By adding STM、SEM experiments and SNP、InDel mutation site analysis, combining the method of polyphasic taxonomy, the content of the paper was enriched and the logic was smoother.

Comment 5: The figures in the article is not clear, it needs to be redrawn by increasing the font.

Response 5: It is really true as Reviewer suggested that figures in the article is not clear. Therefore, we have made appropriate adjustments to the image.

Comment 6: The paper requires major modification.

Response 6: Thank you for taking the time to review our manuscript, as you said, there are many problems with this article and your suggestions are crucial to the revision of the manuscript. Therefore, in the process of rewriting the article, we consider your comments to enrich the article. We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

Reply to Reviewer #2

We are very grateful to your comments for the manuscript. According with your advice, we tried our best to amend the relevant part and made some changes in the manuscript. These changes will not influence the content and framework of the paper. All of your questions were answered below.

Comments: “The authors describe their findings on a Desmodesmus species from Tibetan Yamdrok Lake in Tibet, China via a series of methods including strain isolation, molecular identification and determination of culture conditions. Totally speaking, the authors should clearly emphasize the novelty of this finding in the manuscript. Besides, the following comments are also for your references to improve the quality.”

.1. ‘sp.’ should not in italic form.

2. The resolution of figures is too low to provide information.

3. The data should be carried out in triplicate, and error bars should be provided.

4. In the M&M section, the general process of experimental methods should be concise by referring the published paper.

5. The initial value of TN in Figure 3c should be 200-260 ppm. Please check.

6. There is almost no discussion within the manuscript. The authors should emphasize why they perform this work. What is the significance?

7. The format of the references needs to be unified.

8. There are spelling, grammar and formatting errors in this article, and it should be applied to a professional editing service for language improvement.

Comment 1: ‘sp.’ should not in italic form.

Response 1: Thank you for the detailed review. We have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. We rewritten the article according to the review comments. Editage for its linguistic assistance during the preparation of this manuscript.

Comment 2: The resolution of figures is too low to provide information.

Response 2: Thank you for your valuable advice. It is really true as Reviewer suggested that figures in the article is not clear. Therefore, we have made appropriate adjustments to the image.

Comment 3: The data should be carried out in triplicate, and error bars should be provided.

Response 3: We are very sorry for our negligence of describe. In fact, we used 3 biological replicates, and added to the Methods section of the manuscript，and we have provided error bars in the figure.

Comment 4: In the M&M section, the general process of experimental methods should be concise by referring the published paper.

Response 4: It is really true as Reviewer suggested that referring the published paper in the article is missing. We have improved the M&M section.

Comment 5: The initial value of TN in Figure 3c should be 200-260 ppm. Please check.

Response 5: Thank you very much for your professional review. I'm very sorry that the initial total nitrogen value was not shown on the way during the mapping process. We made corrections. See the picture for details.

Comment 6: There is almost no discussion within the manuscript. The authors should emphasize why they perform this work. What is the significance?

Response 6: Thank you for the detailed review. We carefully modified the methods, results, discussion and conclusion sections. By adding STM、SEM experiments and SNP、InDel mutation site analysis, combining the method of polyphasic taxonomy, the content of the paper was enriched and the logic was smoother.

Comment 7: The format of the references needs to be unified.

Response 7: Thank you for your valuable advice, which will make the revision of the article structure more specific. We rewrote the references section according to the requirements of the journal.

Comment 8: There are spelling, grammar and formatting errors in this article, and it should be applied to a professional editing service for language improvement.

Response 8: Thank you for the detailed review. We have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. We rewritten the article according to the review comments. Editage for its linguistic assistance during the preparation of this manuscript.

I wish this revision will be acceptable for publication in your journal.

Thank you for your consideration. I am looking forward to hearing from you.

Yours Sincerely,

Wang Jinhu

Address: Lhasa, China.

Email: phudor@vip.163.com

Tel: +8613618465558

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

26 Sep 2022

A new Desmodesmus sp. from the Tibetan Yamdrok Lake

PONE-D-22-12548R1

Dear Dr. Duo Bu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Vandana Vinayak, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: