Cultivation of microalgae using flash hydrolysis nutrient recycle

Abstract

Algal biofuels have the potential to provide a scalable source of renewable fuels. The high nutrient use in algae cultivation and its recovery and recycle is one of the challenges that may limit the scalability and sustainability of algal biofuels. The present study evaluates the use of hydrolysate obtained after Flash hydrolysis (FH) of Scenedesmus at 280 °C as a nutrient source for microalgae cultivation. FH hydrolysate nutrient recycling was compared with low temperature batch Hydrothermal Liquefaction (HTL) nutrient recycling. Oocystis and Scenedesmus were cultivated using hydrolysate as a partial phosphorous (P) and nitrogen (N) source. The study shows that hydrolysate from FH could satisfy 50% of the nitrogen and phosphorus demand in the culture media. Ammonia toxicity was identified as one potential limitation to higher percentages of N replacement using HTL aqueous phase as it resulted in a high soluble ammonia concentration in the media. This effect was most pronounced in the 50% P HTL replacement treatment where minimal algae growth was observed during the first 11 days of cultivation.

Introduction

Microalgae contain a mixture of organic and inorganic compounds with the major elements being carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, sodium and other elements present in minor quantities. From a biofuels perspective, the carbon and hydrogen content are the most important since they will provide the majority of the energy yield. From an environmental and scalability point of view, other elements (particularly nitrogen and phosphorus) rise in significance. These nutrients require thoughtful sourcing and as such, the potential for commercial-scale cultivation would benefit tremendously if they could be recovered and recycled from associated waste streams in biologically available forms [1]. Nitrogen is biologically available in the form of ammonium (NH₄⁺) and nitrate (NO₃⁻). Phosphorus is biologically available in the form of phosphate (PO₄^3 −) [2], [3], [4]. Most nitrogen containing fertilizers are produced by reacting atmospheric nitrogen in a process that demands significant amounts of fossil fuels (mainly natural gas as CH₄). The Haber–Bosch process first produces anhydrous ammonia (NH₃) that is then further upgraded [3], [5]. Phosphorus is a mined resource that cannot be substituted and, as a fundamental component in all living organisms, is a crucial element in both the production of biofuels and the food chain [6], [7].

One of the first studies of nutrient recycling in microalgae processing cultured Chlorella vulgaris in the recovered solution after low temperature catalytic gasification; a process by which methane rich fuel gas was obtained. The study concluded that C. vulgaris could grow in the recovered solution, but mixing with a standard media to provide nutrients such as phosphorus and micronutrients was necessary [8]. Biller et al. recently reported the use of recycled aqueous phase (AP) from Hydrothermal Liquefaction (HTL) at two different temperatures (300 and 350 °C) to grow four different algae strains (C. vulgaris, Scenedesmus dimorphus, Spirulina platensis and the cyanobacteria Chlorogloeopsis fritschii). The AP was diluted 50 ×, 100 × and 400 × due to the high concentration of nutrients in it compared to the standard media used to grow the algae strains and to avoid the effects of growth inhibitors such as phenols, fatty acids and nickel (from the reactor wall corrosion). All of the algae strains studied were able to grow in the recycled water, however different optimum dilutions were observed for each [9].

The cultivation of Desmodesmus sp. using AP recovered after HTL of the same microalgae biomass was studied by Garcia-Alba et al. [10] to evaluate the potential of nutrient recycling. HTL experiments were conducted in a batch reactor at 300 °C with 5 min reaction time (excluding pre-heating). The study concluded that when using only AP recovered after HTL diluted with deionized water, a substantial reduction in growth was observed. When a mixture of growth media (COMBO), DI water and AP was used, a growth rate comparable to that observed in COMBO medium alone was reported. The most likely cause for this difference was the lack of essential micronutrients that must be supplied in order to balance the media and optimize the nutrient recycling after HTL [10]. Further studies by Garcia-Alba et al. indicated that the nutrients could be recycled up to five consecutive times using HTL AP [7]. This allowed for recycling of 65.7% of the phosphorous and 39.5% of the nitrogen present in the COMBO medium. Unfortunately, the cell morphology started to change by the fifth recycle. The authors pointed out that aromatic heterocycles were present in the HTL AP and the cell morphology mirrored a study testing the impact of triazines on Scenedesmus sp.[11]. The authors also pointed out that the amounts of potential inhibitory compounds (pyrrolidones, phenols, cyclic dipeptides) were increasing with each successive recycle and that supercritical water gasification of the HTL AP before recycle may be necessary to reduce these compounds to ammonia and CO₂[7].

Lopez Barreiro et al. recently reported that supercritical water gasification of HTL AP greatly reduces the ratio of organic nitrogen to ammonia/ammonium. They also found that up to 75% of the nutrients (using a nitrogen based calculation) present in COMBO and Guillards f/2 media can successfully be recycled depending on algae species [12]. The authors further studied pH variations of the COMBO media by adjusting the final pH of the media to 7.8 (as recommended) and by not doing so (pH = 9.0). The authors proposed that the decline in growth for the high pH treatments was most likely due to ammonium shifting toward ammonia in equilibrium at levels toxic to the algae. Interestingly, for Scenedesmus almeriensis, pH adjustment and supercritical water gasification of the HTL AP did not result in algae growth as extensive as the COMBO media alone. It was also reported that most treatments ran out of total nitrogen (TN) before the 14 day experimental period was over, suggesting that a more nutrient replete medium should be developed and used to allow for longer cultivation periods [12].

Rapid Hydrothermal Liquefaction (R-HTL) of microalgal biomass is a promising subcritical water-based continuous flow conversion and extraction method capable of solubilizing > 60% of the protein present in the biomass and recovering the degraded protein fraction in the hydrolysate as organic nitrogen and its derivatives (a mix of ammonia, amino acids and soluble peptides) [13], [14]. Similarly, > 90% of the organic phosphorus from the microalgal biomass is recovered as soluble orthophosphates along with most of the micronutrients. The short (9 s) residence time of the R-HTL process allows for minimum formation of toxic compounds such as aromatic heterocycles that could potentially negatively impact algae growth [14], [15]. Most of the organic nitrogen present is in the form of soluble peptides and amino acids with small amounts of ammonia. If the algae cannot use the organic nitrogen directly, it will most likely degrade to ammonia over time; especially in the high pH conditions present during algae photosynthesis. This would potentially allow for the slow release of ammonia to the algae [16], [17], [18], [19]; potentially allowing for a continuous low level presence of ammonia that would be beneficial to the algae [20], [21]. Subsequent discussion in this paper regarding R-HTL will be referred to as Flash hydrolysis (FH) due to the use of the FH terminology in prior publications [13], [14].

The objective of the current study was to evaluate the potential use of the hydrolysate obtained after FH as a source of nutrients for a sustainable microalgae production system. The cycling of nitrogen and phosphorus back into the algae culture is expected to reduce the requirements of those nutrients in the balanced cultivation media, therefore resulting in a reduction in the total production cost of algal biomass. In order to compare FH hydrolysate's potential use in nutrient recycling with the established use of HTL AP, batch HTL experiments were conducted using a common reported residence time (30 min) and the same temperature used for FH experiments (280 °C). These conditions were derived from values reported in the literature that included residence times of 5 to 120 min and temperatures from 280 to 375 °C [22]. The robustness of the nutrient recycle process was further compared across the different genera; Scenedesmus and Oocystis.