Comprehensive evaluation of a cost-effective method of culturing Chlorella pyrenoidosa with unsterilized piggery wastewater for biofuel production

Abstract

Background: The utilization of Chlorella for the dual goals of biofuel production and wastewater nutrient removal is highly attractive. Moreover, this technology combined with flue gas (rich in CO₂) cleaning is considered to be an effective way of improving biofuel production. However, the sterilization of wastewater is an energy-consuming step. This study aimed to comprehensively evaluate a cost-effective method of culturing Chlorella pyrenoidosa in unsterilized piggery wastewater for biofuel production by sparging air or simulated flue gas, including algal biomass production, lipid production, nutrient removal rate and the mutual effects between algae and other microbes.

Results: The average biomass productivity of C. pyrenoidosa reached 0.11 g L^-1 day^-1/0.15 g L^-1 day^-1 and the average lipid productivity reached 19.3 mg L^-1 day^-1/30.0 mg L^-1 day^-1 when sparging air or simulated flue gas, respectively. This method achieved fairish nutrient removal efficiency with respect to chemical oxygen demand (43.9%/55.1% when sparging air and simulated flue gas, respectively), ammonia (98.7%/100% when sparging air and simulated flue gas, respectively), total nitrogen (38.6%/51.9% when sparging air or simulated flue gas, respectively) and total phosphorus (42.8%/60.5% when sparging air or simulated flue gas, respectively). Culturing C. pyrenoidosa strongly influenced the microbial community in piggery wastewater. In particular, culturing C. pyrenoidosa enriched the abundance of the obligate parasite Vampirovibrionales, which can result in the death of Chlorella.

Conclusion: The study provided a comprehensive evaluation of culturing C. pyrenoidosa in unsterilized piggery wastewater for biofuel production. The results indicated that this cost-effective method is feasible but has considerable room for improving. More importantly, this study elucidated the mutual effects between algae and other microbes. In particular, a detrimental effect of the obligate parasite Vampirovibrionales on algal biomass and lipid production was found.

Keywords: Biofuel; Chlorella pyrenoidosa; Nutrient removal; Unsterilized piggery wastewater; Vampirovibrionales.

Fig. 1

Algal biomass and lipid production of C. pyrenoidosa in unsterilized piggery wastewater when sparging air or simulated flue gas. The cultures were illuminated at 28 ± 0.5 °C under a 16-/8-h light/dark cycle with exposure to 45 μE m⁻² s⁻¹ provided by cool-white fluorescent lights. The microalgal cells were sampled every 24 h for growth determinations. PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean

Fig. 2

Chemical oxygen demand (COD), ammonium (NH₄⁺-N), total nitrogen (TN) and total phosphate (TP) removal rates. CA means sparging air, CC sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean. **Indicates that there was an extremely significant difference with P < 0.01

Fig. 3

The absolute bacterial abundance based on 16S rRNA copies in unsterilized piggery wastewater. CA means sparging air, CC means sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean. The different letters indicate that there was a significant difference with P < 0.05

Fig. 4

The boxplots of alpha indices (Chao1 and ACE). CA means sparging air, CC sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. *Indicates that there was a significant difference with P < 0.05. The statistical test used to compare the indices was the Wilcoxon signed-rank test

Fig. 5

LEfSe analysis identified the most differentially abundant taxa between CA and PA. The taxonomic cladogram was obtained from LEfSe analysis of 16S rRNA sequences; only taxa meeting an LDA significance threshold of 4.0 are shown. Small circles and shading with different colors in the diagram represent the abundance of those taxa in the respective group. Yellow circles represent nonsignificant differences in abundance between CA and PA for that particular taxonomic group. The brightness of each dot is proportional to its effect size. Taxa enriched in PA are shown with a positive LDA score (green) and taxa enriched in CA have a negative score (red). CA means sparging air; PA means culturing C. pyrenoidosa with sparging air

Fig. 6

LEfSe analysis identified the most differentially abundant taxa between CC and PC. The taxonomic cladogram was obtained from LEfSe analysis of 16S rRNA sequences; only taxa meeting an LDA significance threshold of 4.0 are shown. Small circles and shading with different colors in the diagram represent the abundance of those taxa in the respective group. Yellow circles represent nonsignificant differences in abundance between CC and PC for that particular taxonomic group. The brightness of each dot is proportional to its effect size. Taxa enriched in PC are shown with a positive LDA score (green), and taxa enriched in CC have a negative score (red). CC means sparging simulated flue gas; PC means culturing C. pyrenoidosa with sparging simulated flue gas