DOI: 10.5281/zenodo.15438118
title: The dataset for the study titled "Flow-through and cyclic fed-batch cultivation of the green microalga Haematococcus pluvialis in recirculating aquaculture effluent: Strategies for sustained biomass production and astaxanthin biosynthesis"

description: This dataset is associated with the study titled "Flow-through and cyclic fed-batch cultivation of the green microalga Haematococcus pluvialis in recirculating aquaculture effluent: Strategies for sustained biomass production and astaxanthin biosynthesis," published in Aquacultural Engineering.

Authors: Hemanta Timilsina1*, Minna Hiltunen1, Marco L. Calderini2, Pauliina Salmi2, Juhani Pirhonen1, Katja Pulkkinen1

1Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FI-40014, Jyväskylä, Finland 2Faculty of Information Technology, University of Jyväskylä, P.O. Box 35, FI-40014, Jyväskylä, Finland

*Corresponding author email: hemanta.h.timilsina@jyu.fi

The dataset file consists of the following file:timilsina_etal_Hpluvialis_data

This file contains data from two separate lab-scale experiments (Exp I and II) investigating the cultivation of the microalga Haematococcus pluvialis in recirculating aquaculture system (RAS) effluent under flow-through and cyclic fed-batch cultivation systems. The study evaluated growth parameters (cell density, growth rate, dry weight, biomass productivity, and chlorophyll-a), nutrient (total N, NO3-N, and PO4-P) removal efficiency, biochemical composition (carbon and nitrogen percentage of dry weight), and astaxanthin content.

Materials and methods

For detailed methodologies, refer to the full article.

Briefly, H. pluvialis (strain K-0084 (NIVA), Sweden) was cultivated under flow-through and cyclic fed-batch cultivation systems to assess growth parameters (cell density, dry weight, biomass productivity, and chlorophyll-a content), nutrient removal efficiency, and astaxanthin biosynthesis. Exp I compared growth in a synthetic culture medium (modified BG-11) and untreated recirculating aquaculture system (RAS) effluent, while Exp II evaluated the effects of autoclaved and filtered-autoclaved RAS effluent. Both experiments employed quadruplicate (n=4) treatments per cultivation mode. Flow-through cultivation was conducted in chemostats (Laborexin Oy, Finland) with a total volume of 455 mL and a working volume of 440 mL, whereas cyclic fed-batch cultivation was conducted in repurposed transparent bottles (1500 mL) with a working volume of 880 mL. Illumination was provided by 36 W/865 cool daylight fluorescent lamps (Osram, Poland) under an 18:6 h light-dark photoperiod, with intensities of 30–40 µmol m⁻²s⁻¹ in Exp I and 45–55 µmol m⁻²s⁻¹ in Exp II. Light stress (LED, 18W, AP67 T8 tubes, Valoya Oy, Finland; 200–250 µmol m⁻²s⁻¹) was applied to the outflow from flow-through cultivation and the withdrawn culture from cyclic fed-batch cultivation to induce astaxanthin biosynthesis (Exp II only).

Cell density was quantified daily using a hemocytometer (Bürker, Marienfeldt) under a 100x magnification under a microscope. Dry weight (DW) was assessed by filtering a known culture volume through a pre-weighed glass fiber filter (Whatman GF/C), followed by drying and weighing. Chlorophyll-a (chl-a) content was determined via ethanol extraction and spectrophotometric analysis (UV-1800, Shimadzu, Japan). DW and chl-a measurements were taken at the late-exponential phase and the final day of cultivation.

Total nitrogen (TN), nitrate (NO3-N), and phosphate (PO4-P) were measured at three time points: (i) before inoculation, (ii) at the late-exponential phase, and (iii) on the final cultivation day. NO3-N and PO4-P were quantified using reagent kits (LCK349 and LCK339, Hach, USA) with a spectrometer (LASA 100, Dr. Lange, Germany). TN was measured with a TOC/TN analyzer (TOC-L CSH/CSN, Shimadzu, Japan). Nutrient removal efficiency was calculated as the percentage reduction from the initial concentration in the medium.

In Exp II, algal biomass (green- and red-phase) was harvested via centrifugation, frozen, lyophilized for C and N (% DW) analysis. Ca. 0.9 mg of green and red biomass samples was analyzed using a Thermo Finnigan DELTAplus Advantage continuous flow stable isotope-ratio mass spectrometer (CF-SIRMS) connected to a Carlo Erba Flash EA1112 elemental analyzer (Thermo Scientific, Germany).

In Exp II, outflow from flow-through cultivation and withdrawals from cyclic fed cultivation were subjected to light stress with aeration to induce astaxanthin biosynthesis, with samples collected at multiple time points. Stressed cells were centrifuged, cryopreserved at −80°C, and lyophilized. Astaxanthin was extracted using acetone with an internal standard, de-esterified via saponification, purified, concentrated, and analyzed by High-Performance Liquid Chromatography (Nexera HPLC, Shimadzu, Japan) with a Photodiode Array Detector (SPD-M20A).

Each treatment consisted of four replicates (n = 4), including modified BG-11 and U-RAS in Exp I, and A-RAS and FA-RAS in Exp II. All parameters were analyzed using n = 4, except for the U-RAS treatment under flow-through cultivation in Exp I, where one replicate was excluded from DW, chl-a, and nutrient removal analyses due to technical issues, specifically, sample loss during handling and processing. These parameters were therefore analyzed using n=3.