Biochemical and phylogenetic characterization of the wastewater tolerant Chlamydomonas biconvexa Embrapa|LBA40 strain cultivated in palm oil mill effluent

Abstract

The increasing demand for water, food and energy poses challenges for the world´s sustainability. Tropical palm oil is currently the major source of vegetable oil worldwide with a production that exceeds 55 million tons per year, while generating over 200 million tons of palm oil mill effluent (POME). It could potentially be used as a substrate for production of microalgal biomass though. In this study, the microalgal strain Chlamydomonas biconvexa Embrapa|LBA40, originally isolated from a sugarcane vinasse stabilization pond, was selected among 17 strains tested for growth in POME retrieved from anaerobic ponds of a palm oil industrial plant located within the Amazon rainforest region. During cultivation in POME, C. biconvexa Embrapa|LBA40 biomass productivity reached 190.60 mgDW • L-1 • d-1 using 15L airlift flat plate photobioreactors. Carbohydrates comprised the major fraction of algal biomass (31.96%), while the lipidic fraction reached up to 11.3% of dry mass. Reductions of 99% in ammonium and nitrite, as well as 98% reduction in phosphate present in POME were detected after 5 days of algal cultivation. This suggests that the aerobic pond stage, usually used in palm oil industrial plants to reduce POME inorganic load, could be substituted by high rate photobioreactors, significantly reducing the time and area requirements for wastewater treatment. In addition, the complete mitochondrial genome of C. biconvexa Embrapa|LBA40 strain was sequenced, revealing a compact mitogenome, with 15.98 kb in size, a total of 14 genes, of which 9 are protein coding genes. Phylogenetic analysis confirmed the strain taxonomic status within the Chlamydomonas genus, opening up opportunities for future genetic modification and molecular breeding programs in these species.

Fig 1. Representation of Palm Oil Mill Effluent (POME) wastewater treatment ponds (DENPASA—Dendê do Pará S/A).

Fig 2. Screening of microalgae strains for growth in anaerobic pond palm oil mill effluent (AP-POME): Screening was carried out in 500 ml erlenmeyer flasks containing 250 ml of AP-POME as culture medium.

Batch culturing was independently conducted in triplicates for each of the 17 strains at a constant aeration of 5 L·h^-1of atmospheric air, at 26 ± 1°C, light intensity of 100 μEm^-2 s^-1 (7500 lux), and 12/12h light/dark cycle. Microalgal growth was monitored through periodic measures of absorbance at 680nm and microscopic inspection of culture samples during 10 days of cultivation. The absorbance of AP-POME prior to algal inoculation was used as blank. Initial and final absorbances were used to calculate basal growth rate (μ). *Results are presented as mean ± error bars of triplicate experiments (n = 3).

Fig 3. Growth dynamics of Chlamydomonas biconvexa Embrapa|LBA40 under different conditions.

The strain was cultivated in erlenmeyer flasks using 250 mL of AP-POME or BBM (control) under different conditions: Axenic culture using BBM and 12/12 h light/dark cycles (−□−), axenic culture using undiluted AP-POME in the light (−■−); axenic culture using undiluted AP-POME in the dark (−●−), non-axenic culture using undiluted AP-POME and 12/12 h light/dark cycles (··■··), non-axenic culturing using 50% diluted AP-POME in distillate water and 12/12 h light/dark cycles (··■··). Batch cultures were aerated with 5 L·h^-1 of atmospheric air, at 26°C ± 1°C, at light intensity of 100 μEm^-2 s^-1 (7500 lux) (when applicable). The growth kinetics of the algae was evaluated through cell counting using a Neubauer chamber. Results shown are the mean of biological triplicates of the experiment (n = 3).

Fig 4. Growth dynamics of Chlamydomonas biconvexaEmbrapa|LBA40 in airlift flat plate photobioreactors.

The strain was batch cultivated using in airlift flat plate photobioreactors under non-axenic conditions. The working volume of 13 L of cultivation medium, bold basal medium (BBM) (··○··) or anaerobic pond palm oil mill effluent (AP-POME) (··●··), was used with constant aeration of 60 L·h-1 and CO2 supplementation adjusted to 5% of the air flow. Experimentation was conducted at 12h/12h light/dark cycling regimen, at a light intensity of 495 μEm^-2 s^-1 (35000 Lux) and temperature of 25° C during dark periods and 35° C during the light periods. Biomass dry weight (DW) was gravimetrically determined using 10 ml samples retrieved from cultures from each photobioreactor replicate at the initial time and at 5 days intervals during 15 days of culturing. Results shown are the mean of biological triplicates of the experiment (n = 3).

Fig 5. Representation and comparison of the mtDNA from Chlamydomonas biconvexaEmbrapa|LBA40 strain and mtDNA from the green microalgae reference Chlamydomonas reinhardtii [32].

Genes: nad1—Subunit 1 NADH dehydrogenase fragmented; rnL7, rrnL8—Large subunit ribosomal RNA; trnQ-UUG—Transfer RNA, initiation codon amino acid tryptophan; rrnS1, rrnS3, rrnS4—Small subunit ribosomal RNA; trnW-CCA—Transfer RNA, initiation codon amino acid glutamine; nad6—Subunit 6 NADH dehydrogenase; nad2—Subunit 2 NADH dehydrogenase; cox1—Cytochrome c oxidase subunit I; cob—Apocytochrome b protein; nad5—Subunit 5 NADH dehydrogenase; nad4—Subunit 4 NADH dehydrogenase; trnM—Transfer RNA, initiation codon amino acid methionine; nad1—Subunit 1 NADH dehydrogenase.

Fig 6. Phylogenetic tree based on mitochondrial cox1 gene sequence inferred by using Maximum Likelihood method and JTT matrix-based model.

The bootstrap consensus tree inferred from 100 replicates. Analysis involved amino acid cox 1 gene sequences from 30 microalgae strains and were conducted in MEGA X.