Production of Oleaginous Microbial Biomass by Reusing Wastewaters

Abstract

Global energy demand continues to increase, which raises the question regarding how to solve the energy crisis caused by diminishing fossil fuels. There is no single alternative energy source that could substitute the fossil fuels, but microbial single cell oils (SCO) could be part of the solution. SCOs can be produced by cultivating microorganisms in wastewater in which nutrients and carbon from the wastewater are used for biomass production. In optimized conditions, microorganisms begin to accumulate lipids, and these lipids can be further refined for the production of biodiesel or renewable diesel. The lipid accumulation of the microorganisms may be enhanced by culturing the microorganisms under stressful conditions. The most commonly used strategy for enhancing lipid accumulation is nitrogen starvation, but it is even more effective when combined with another stress factor, such as moderately increased salinity.

In microbial lipid production, the major cost factor is often the substrate needed for the microorganisms. Therefore, utilizing inexpensive substrates and waste materials for the cultivation of oleaginous microorganisms is very desirable. Various wastewaters from municipalities, agriculture, and industrial sources have been studied, and many of these wastewaters have shown the potential for lipid-rich biomass production. Unfortunately, most of the studies have been conducted using sterilized wastewater. In large-scale applications, the sterilization of the wastewater is not cost-effective; therefore, lipid-accumulating microorganisms able to compete with the indigenous microorganisms of the wastewater need to be further studied.

The aim of this work was to sustainably produce oleaginous biomass by reusing the carbon and nutrients from wastewaters. This work included an evaluation of the suitability of various wastewaters for lipid-lipid rich biomass production (Paper I), the isolation of yeasts and fungi, which could possibly accumulate lipids by utilizing wastewater as substrate (Paper II), and the determination of the ability of the isolated microorganisms to accumulate lipids by comparing them with known lipid accumulating yeasts (Paper II). Unlike yeasts and fungi, microalgae are able to use an inorganic carbon source for their growth. This feature enables the combination of wastewater and flue gas treatment. Therefore, the growth and lipid accumulation of three microalgal species were compared (Paper III), and the suitability of the most potential microalgal species for accumulating lipids in sterilized and non-sterilized wastewater was studied (Paper III & IV).

Based on the results of this study, palm oil mill effluent (POME) has more potential for lipid production than chemithermomechanical pulp mill effluent (CTMP) or municipal wastewate r (MWW) (Paper I). The residual lipids and solids of POME obstructed the analyses of the microbial SCOs.

Eukaryotes isolated from POME with agar plates were genetically identified as Candida silvae NRRL Y-6725 (with 100% similarity), Galactomyces geotrichum LMA-20 (with 99.8% similarity), Lecythophora hoffmannii CBS245.38T (with 96.7% similarity), and Graphium penicillioides JCM9300 (with 99.3% similarity) (Paper II). The fungus Graphium penicillioides had a great potential for lipid accumulation based on the comparison study with well-known oleaginous yeast strains (Yarrowia lipolytica DSMZ8212, Cryptococcus curvatus DSMZ70022, & Cryptococcus albidus DSMZ701097) in a synthetic medium (Paper II). The lipid content per dry weight was higher with G. penicillioides compared to C. curvatus after 15 days of incubation (29.1±3.0 wt% vs 20.2±2.9 wt%, Paper II). Unfortunately, the overall lipid concentration was lower due to a lower biomass concentration. G. penicillioides contained more than 20% lipids, so it can be called oleaginous.

From the three microalgae isolated from a Taiwanese freshwater area (Chlorella sorokiniana CY1, Chlorella vulgaris CY5, & Chlamydomonas sp. JSC-04), C. vulgaris accumulated more lipids when various media, nitrogen sources, and nitrogen concentrations were studied (Paper III). The C. vulgaris in the BG-11 medium, initially containing 0.38 g NaNO3/L, produced 3.8 g/L biomass and 57.5 wt% lipids after 12 days of incubation. The most suitable wastewater dilution for the lipid accumulation of C. vulgaris on sterilized anaerobically treated piggery wastewater was 5x dilution, which resulted in initial chemical oxygen demand and total Kjeldahl nitrogen of 75.4 mg/L and 57.4 mg/L, respectively. C. vulgaris was suitable for accumulating lipids on both sterilized and non-sterilized anaerobically treated piggery wastewater (PW) (Paper IV). The highest lipid content and productivity with the non-sterilized wastewater were rather promising (32.5±3.2 wt%, 71.2±2.2 g/L/d). However, under the conditions of these experiments, C. vulgaris excreted dissolved organic carbon (Paper III & IV), and the aim in wastewater treatment is the removal of organic carbon.

In summary, this work demonstrates the potential of indigenous eukaryotic microorganisms for lipid-rich biomass production. G. penicillioides isolated from POME has the potential for lipid-rich biomass production in a synthetic medium, which has not been previously reported. Similarly, C. vulgaris has the potential for lipid-rich biomass production in non-sterilized piggery wastewater, while most of the studies in the literature on C. vulgaris and wastewater have been conducted using sterilized wastewater. To enable simultaneous accumulation of lipids and efficient treatment of wastewater, special attention should be focused on the growth conditions.