Biofuel production, digestate purification and CO2 sequestration

1. Auto-floating microalgae: selection and cultivation

The BIO-RPISM research team have developed a novel technology (patent: PCT/EP2021/066405) for selecting and cultivating auto-floating microalgae, and the use of such microalgae for wastewater bioremediation, CO2 sequestration, and biofuels/bioproducts production. The auto-floating microalgae can effectively float at the water surface without the need for flocculants and flotation gas, which offers a significant saving in capital investment over other recognized methods.

Selective process leading to the enrichment of auto-floating mciroalgae from mixed algal culture
Autoflotation (in 30 min, 28x fast forward) without cogulation and external flotation gas supply

According to this method, a highly productive filamentous microalga (Tribonema sp.) has been derived from mixed algal cultures naturally developed in wastewater. The microalga demonstrates several specific qualities including effective autoflotation (ca. 80-95%), excellent dewaterability (solids content to ca. 20% via direct microfiltration), high lipid productivity (ca. 70 mg/L/d), high carbohydrate content (40-60% of dry cell weight), and excellent bioremediation performance (nutrient removal nearly 100%). These characteristics make it a promising feedstock for biofuel and high-value algal product production. For example, by coupling auto-floating microalgae cultivation with anaerobic digestion, we have demonstrated a closed loop system for simultaneous biofuel production, digestate purification, and CO2 sequestration (SEAI 18/RDD/299 project). The microalga also has a high beta-1-3/1-6-glucans (ca. 60% of total carbohydrates) content. Beta-1-3/1-6-glucans are known to have many biological activities (e.g., anti-oxidation, anti-tumour, anti-inflammatory, and immune-modulating properties, etc.) and have wide applications in pharmaceuticals, nutraceuticals, food, feed, and cosmetics sectors. Based on these characteristics, we acquired an EI Commercialisation Case Feasibility grant, led one Horizontal Europe application, and participated in another Horizontal Europe application. We have also joined the BIM Aquatech Innovation Studio to develop novel microalgae-based aquaculture systems.

2. Growth and lipids accumulation characteristics of auto-floating microalgae in digestate

A series of tests were conducted to investigate: (1) the biodiesel production potential of the auto-floating microalgae (Tribonema sp.); (2) optimal CO2 concertation; and (3) effect of digestate loading (based on NH4-N) on growth and biodiesel production.

The results show that the auto-floating microalgae have a high biodiesel potential, with a total lipids content of 42%, 29% and 34% in BG11, digestate and secondary wastewater respectively (Fig. 1a). However, the growth was restricted in digestate with 2% CO2 (Fig. 1b). The microalgae grew well in digestate at 5% CO2 with high biomass productivity, while the growth was hampered at both low (2%) and high (15%) CO2 concentrations (Fig. 1c). Five percent is a typical CO2 concentration in biogas-fuelled CHP exhaust, which validates that auto-floating microalgae cultivation can be integrated with biogas production and utilization.

Fig. 1 Biodiesel potential of auto-floating microalgae (at 2% CO2) and optimal CO2 concertation for growth in digestate: (a) cellular composition at the end of test (day 21); (b) growth characteristics with different growth medium; and (c) effect of CO2 concertation on growth of auto-floating microalgae in digestate

At the optimal CO2 concentration (5%), the auto-floating microalgae attained high biodiesel productivity at NH4-N concentrations of 100-250 mg/L (Fig. 2). The algae started to accumulate lipids from the late logistic phase (day 12). The results show that low digestate loading (NH4-N=50 mg/L) leads to the highest biodiesel yield, but the finial productivity is limited due to nutrient limitation. High digestate loading (NH4-N≥375 mg/L) results in growth inhibition. Although all the objectives of WP1 have been met, further study is underway to investigate the inhibitory factors and enhancement strategies for high digestate loadings.

Fig. 2 Effects of digestate loading on growth and biodiesel production: (a) growth curve; (b) FAMEs yield (of dry cell weight, DCW); and (c) biodiesel productivity

3. Long-term stability of the auto-floating microalgae grown in digestate/centrate

Long-term semi-continuous experiment over 203 days has been conducted using a laboratory scale column photo-SBR (6.65 L). The general conditions were: CO2 supply, 5% CO2 at 0.01 vvm; light/dark periods, 10/14 hours; light intensity, ca. 350 µmol/m2/s; volume exchange ratio, 0.7; separation time, 1 h. Based on different hydraulic retention time (HRT) and cell retention time (CRT), three operational scenarios were tested: (1) HRT, 1.4 d, CRT, 5d; (2) HRT 2.8 d, CRT 10 d; (3) HRT 4.2 d, CRT 5 d. The results (Fig. 1) show that long-term stability (over 160 days) of the auto-floating microalgae could be maintained under mixed culture condition, with an average autoflotation efficiency of >75%. Then, the autoflotation deteriorated and the culture was contaminated by unicellular green algae. This was mainly caused by the variation of cetrarate, i.e., deficiency of some key growth elements (phosphorus and sulphate). Therefore, these factors should be regularly monitored and supplemented if necessary.

Long-term stability of of auto-floating microalgae grown in digestate/centrate

Cellular compositions under different operational conditions are summarized in the table below. Although the microalgae did not attain high lipid contents, it achieved high carbohydrate contents in the range of 33.3-51.5%. Microalgal carbohydrate is an ideal feedstock for the production several renewable biofuels including biogas, bioethanol, biobutanol and bio-crude oil. Overall, the results suggest that cultivation of auto-floating microalgae in digestate/centrate is a feasible and promising technical route for integrated wastewater purification, CO2 sequestration, and biofuel production.

Cellular compositions under different operational conditions

4. Auto-floating microalgae as an advantageous co-substratesubstrate for enhanced methane production

Anaerobic digestion is one of the most effective pig manure management practices owing to its multiple benefits including bioenergy (methane-rich biogas) generation, GHG mitigation, pathogens reduction, and its flexibility in different scales. However, due to the high nitrogen content of pig manure, its anaerobic digestion often suffers ammonia inhibition and subsequent low bioenergy conversion efficiency. To overcome this issue, co-digestion of pig manure with carbon-rich substrates has been intensively investigated as the most feasible option. Various biomass and organic wastes, such as energy crops, agricultural residue, municipal waste, and industrial waste have been explored as co-substrates for AD of manure. Although enhanced performance is generally obtained, the economic and environmental consequences of anaerobic co-digestion are often not as positive as expected, due to the costs and environmental impacts associated with obtaining and handling the co-substrates. Long-term and stable supply of environmentally friendly co-substrates has been identified as the determinant to ensure the economic viability and environmental performance of pig manure co-digestion.

In recent years, microalgae have been identified as an attractive AD feedstock, owing to its high productivity, significant CO2 mitigation effect, non-competition with arable land and its possibility to integrate with other technological processes such as wastewater purification and biorefinery. A particularly attracting aspect of microalgae as an AD feedstock is that microalgae cultivation can be fully coupled with digestate (AD by-product) valorisation and biogas upgrading, thus closing the loop in microalgal biomass production and AD by-product management. Microalgal biomass from different sources has been demonstrated as efficient co-substrates for enhanced methane (CH4) generation in AD of various substrates including pig manure. Most used microalgae species are unicellular microalgal genera such as Chlorella and Scenedesmus. Despite its advantages, however, there are also some technical restraints in AD of microalgal biomass, including low biomass concentration in the microalgal culture broth, unbalanced carbon to nitrogen (C/N) ratio of microalgal biomass, recalcitrant cellular constituents and the cost of microalgal biomass production (cultivation, harvesting, and dewatering). Among these, the economic and energetic costs associated with harvesting and dewatering of microalgal biomass are major techno-economic hurdles for the practicality of energy production from microalgae.

Auto-floating mciroalgae can form floating mats in non-turbulent water bodies, which can be harvested by simple methods like scrapping. Besides, the high filterability of auto-floating microalgae also renders them good dewaterability. Therefore, the use of auto-floating microalgae could potentially overcome the harvesting and dewatering problems, thus improving the practicability of bioenergy generation from microalgae. In the Bio-RPISM project, we explored auto-floating microalgae (Tribonema sp.) as an advantageous co-substrate for anaerobic digestion (AD) of pig manure. Its impacts on the AD performance were assessed in terms of methane yield, energy conversion efficiency, digestion kinetics, and digestate dewaterability. The microalgae substantially improved methane yield, AD kinetics, and digestate dewaterability of the AD process. The enhancement in methane yield ranged from 2 to 27.4%, with the maximum enhancement (corresponding to an energy conversion efficiency of 81%) occurring at a mixing ratio of 1:1 (VS basis). The AD kinetics was improved as indicated by the increased hydrolysis rate constants and diminished lag time. The specific resistance to filtration (SRF) of the digestate decreased significantly with the increasing proportion of the microalgae in the co-substrates, which would facilitate digestate processing and valorisation. Subsequently, the high biomass productivity of the microalgae (441 mg/L/d) in liquid digestate would enable sustainable bioenergy production through nutrient recycling.