Microalgae and culture conditions

C. vulgaris FACHB-2723 (obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Science, Wuhan, China) was used in this study. The ingredients of BG11 medium used in the experiments are listed in Supplemental Tables S1 and S2. The autotrophic condition was run using 100 mL of medium BG11 in 250-mL flasks illuminated from one side with bubbling of sterilized air containing 5% CO2 (5% CO2-air) at 25 °C. The light source used was 40-W daylight fluorescent lamps at 10 kilolux at the surface of medium. The heterotrophic condition was performed using 100 mL of medium BG11 containing 10 g/L of glucose solution and 50 µg/mL of ampicillin solution (Biofroxx, Einhausen, Germany) in each 250-mL cotton plugged Erlenmeyer flask, on a thermostatic shaker (216 rpm) at 25 °C in the dark. For the photoheterotrophic condition, the illumination and culture process were the same as in the autotrophic condition except that the culture medium BG11 contained 10 g/L of glucose solution and 50 µg/mL of ampicillin solution. Algae in logarithmic growth phase were seeded at a density of 5 ~ 10 × 105 cells/mL into a 250-mL conical flask containing 100-mL culture medium, and then transferred to the corresponding culture conditions. During the culture process, the culture medium was changed every 3 days. All of the experiments were repeated at least 3 times to assure three separate flasks (replicates) analyzed at the same time.

Evaluation of growth performance

Chlorella in the logarithmic growth phase was inoculated at the same density (1.0 × 106 individuals/mL) and cultured for 15 days under the autotrophic condition, heterotrophic condition, or photoheterotrophic condition. The cells were harvested at 24 h intervals until 15 days. The harvested cells were counted by a cell counter (Countstar BioMed, Shanghai, China) in triplicates for each sampling time point, and the average cell count was used to plot the growth curve.

Sample collection

According to the growth curve of C. vulgaris mentioned above, at the 7th day of Chlorella culture, the biomass obtained was harvested by centrifugation at 1700 × g for 10 min, then fast frozen in liquid nitrogen, and stored at − 80 °C for further analyses on the nutrient contents and transcriptome. Based on the growth status of the algae, only Chlorella samples from the autotrophic condition and the photoheterotrophic condition were tested in the transcriptome analysis.

Determination of moisture and protein contents

The moisture content in the Chlorella samples was determined by vacuum freeze-drying method. After dehydration, the total protein content was analyzed using the Kjeldahl method with a nitrogen-to-protein conversion factor of 6.25, as described by the national standard of the People’s Republic of China (GB/T5009.5–2003).

Lipid and fatty acid profile analyses

Total lipid of the frozen-dried Chlorella meal was determined using the chloroform–methanol (1: 2, v/v) extraction method (de Jesus et al. 2018).

After determining the lipid content, the dried residue was methylated with boron trifluoride (BF3) in methanol, and then the methyl esterified samples were analyzed by gas chromatography (Agilent 6890, Santa Clara, CA, USA) with an OmegawaxTM320 column (Agilent 6890, Santa Clara, CA, USA). Chromatography was carried out as described in our recent literature (Zhou et al. 2023). Individual fatty acid methyl ester (FAME) peaks were identified according to their retention times in comparison to the retention times of known FAMEs in a FAME standard (Nu-Chek Prep. Inc, Elysian, MN, USA) containing 40 FAMEs. Identified fatty acids were presented as an area percentage of the total fatty acid (Ördög et al. 2013).

Amino acid analysis

For amino acid analysis, dried algae were ground and 0.1 g samples were hydrolyzed with 10 mL hydrochloric acid (6 M) at 110 °C in microreaction vials under nitrogen for 24 h. Following hydrolyzation and derivatization, the sample was measured by a fully automated amino acid analyzer (Hitachi L-8800, Hitachi Ltd., Tokyo, Japan) for the detection of 16 amino acids, including aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, ammonium chloride, and arginine.

Transcriptome data analysis

Total RNA was extracted from the cells using TRIzol® Reagent according the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) and genomic DNA was removed using DNase I (TaKaRa, Tokyo, Japan). RNA degradation and contamination were monitored on 1% agarose gels. Then the integrity and purity of the total RNA quality were determined by 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA) and the RNA content was quantified using the ND-2000 (NanoDrop Technologies, Waltham, MA, USA). Only a high-quality RNA sample (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 8.0, 28S: 18S ≥ 1.0, > 1 μg) was used to construct sequencing library. The transcriptome library was prepared following a TruSeq TM RNA sample preparation Kit (Illumina, San Diego, CA, USA) using 1 g of total RNA. Shortly, messenger RNA was isolated according to the poly-A selection method by oligo (dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with random hexamer primers (Illumina, Santa, Clara, CA, USA). Then the synthesized cDNA was subjected to end-repair, phosphorylation, and ‘A’ base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low-Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB, Ipswich, MA, USA) for 15 PCR cycles. After quantified by Qubit 4.0 fluorometer (Thermo Scientific, Waltham, MA, USA), paired-end RNA-seq sequencing library was sequenced with the Illumina NovaSeq 6000 sequencer (2 × 150-bp read length; Illumina, San Diego, CA, USA) by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China).

The software fastp (https://github.com/OpenGene/fastp) was used for the adapter trimming and quality control of the raw paired-end reads with default parameters. Briefly, clean reads obtained by removing adapter reads and low-quality reads (quality score < 20) were used to do de-novo assembly with Trinity (http://trinityrnaseq.sourceforge.net/). Then the assembled transcripts were assessed and optimized with BUSCO (Benchmarking Universal Single-Copy Orthologs, http://busco.ezlab.org), TransRate (http://hibberdlab.com/transrate/), and CD-HIT (http://weizhongli-lab.org/cd-hit). All the assembled transcripts were searched against the NCBI protein non-redundant database (NR, ftp://ftp.ncbi.nlm.nih.gov/blast/db/), Swiss-Prot (http://web.expasy.org/docs/swiss-prot_guideline.html), Pfam (http://pfam.xfam.org/), Clusters of Orthologous Groups of proteins (COG, http://www.ncbi.nlm.nih.gov/COG/), GO (Gene Ontology, http://www.geneontologo.org), and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/keeg/) databases using BLASTX to identify the proteins that had the highest sequence similarity with the given transcripts to retrieve their function annotations. A typical cut-off E-value < 1.0 × 10−5 was set.

The differentially expressed genes (DEGs) were obtained by differential analysis software DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/) according to the standards of p-adjust < 0.05 and | log2 (fold change) |≥ 1.5, and the functional enrichment of the DEGs was analyzed by Gene Ontology (GO, http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/).

Quantitative real-time PCR validation

Samples of algal cells grown under the photoheterotrophic and the autotrophic conditions were harvested and were used for qPCR validation. RNA extraction and RNA quality check were conducted as previously described. The Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus, Yeasen, Shanghai, China) was used for reverse transcription of 1 µg of total RNA according to manufacturers’ instructions. The quantitative real-time PCR (qRT-PCR) for gene expression assay was performed using Hieff® qPCR SYBR® Green Master Mix (No Rox) (Yeasen, Shanghai, China) on a quantitative thermal cycler (Light Cycler 480II, Roche, Basel. Switzerland). Specific primer pairs were designed for each target gene through Primer Premier (version 5.0) software (PREMIER Biosoft, San Francisco, CA, USA) using known sequences in the NCBI database (Supplemental Table S3). The qRT-PCR conditions were as follows: preincubation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, annealing temperature (corresponding specific primer pairs) for 20 s, and 72 °C for 20 s. Melting curves were systematically monitored (temperature gradient at 0.5 °C/s from 60 to 95 °C). Using 18S as reference genes, the genes were quantified using the 2−∆∆Ct value method according to Pfaffl (2001). The value of algal cell in the autotrophic condition was assigned as an arbitrary value of 1.

Statistical analysis

All of the experiments were repeated 3 times independently (three separate flasks analyzed at the same time), and data were recorded as the mean with standard deviation (SD). The growth data and nutrient parameters were subjected to one-way ANOVA (analysis of variance) followed by the post hoc (Duncan’s multiple range test) to test the effects of trophic treatment on algal growth and nutrient contents. Differences between genes validated were identified by independent t-test. Statistical analyses were performed using SPSS software (version 26.0, IBM, Armonk, NY, USA), and a p-value < 0.05 was considered statistically significant. The threshold for the DEG identification and the KEGG pathway enrichment were set at p-values < 0.05 corrected by the Benjamini and Hochberg correction method.