The Emissions of a Compression-Ignition Engine Fuelled by a Mixture of Crude Oil and Biodiesel from the Lipids Accumulated in the Waste Glycerol-Fed Culture of Schizochytrium sp.

Abstract

Microalgae are considered to be a promising and prospective source of lipids for the production of biocomponents for conventional liquid fuels. The available sources contain a lot of information about the cultivation of biomass and the amounts and composition of the resulting bio-oils. However, there is a lack of reliable and verified data on the impact of fuel blends based on microalgae biodiesel on the quality of the emitted exhaust gas. Therefore, the main objective of the study was to present the emission characteristics of a compression-ignition engine fuelled with a blend of diesel fuel and biodiesel produced from the lipids accumulated in the biomass of a heterotrophic culture of Schizochytrium sp. The final concentrations of microalgal biomass and lipids in the culture were 140.7 ± 13.9 g/L and 58.2 ± 1.1 g/L, respectively. The composition of fatty acids in the lipid fraction was dominated by decosahexaenoic acid (43.8 ± 2.8%) and palmitic acid (40.4 ± 2.8%). All parameters of the bio-oil met the requirements of the EN 14214 standard. It was found that the use of bio-components allowed lower concentrations of hydrocarbons in the exhaust gas, ranging between 33 ± 2 ppm and 38 ± 7 ppm, depending on the load level of the engine. For smoke opacity, lower emissions were found in the range of 50–100% engine load levels, where the observed content was between 23 ± 4% and 53 ± 8%.

1. Introduction

1.1. Prospects of Biofuels

One of the priorities of researchers and engineers is to develop and then effectively implement clean, efficient, and cost-effective technologies for energy production. This will enable the ecological utilisation of fossil fuels, which will have a direct impact on the reduction of carbon dioxide (CO₂) emissions into the atmosphere. As a consequence, the dynamics of the observed global warming and the catastrophic phenomena triggered by this process will decrease [1]. The dynamic development of renewable energy sources will also enable the stabilisation of market prices for fuels, lead to more innovation, influence technological development, and increase economic growth [2].

Unfortunately, without various types of support programmes and instruments, low-interest loans and credits, as well as tax regulations, the competitiveness of energy systems based on renewable sources is currently low [3]. Previous attempts to implement biofuel production have shown that such plants are characterised by a complicated structure, technological complexity, and operational difficulties, and they require high investment costs, which have direct impacts on their low economic efficiency [4]. Especially when the implementation activities focus on the expected production of third-generation biofuels [5].

However, it should be emphasised that the market situation and public attitudes toward the clean energy sector have changed in recent years. The observed enormous fluctuations and volatility of energy commodity prices on the conventional fuel exchange, related to the complicated geopolitical situation in the world, have led to increased interest in and the profitability of clean energy production [6]. It has been shown that the production of energy sources, the supply of which is relatively constant and at the same time not determined by climatic and atmospheric factors, is justified [7]. These criteria are met by biomass and various types of biofuel, including methane, hydrogen, biohythane, bioethanol, or bio-oil for biodiesel production [8]. The production and proper use of biofuels in the energy mix stabilises energy prices and enables societies without access to fossil fuel resources to achieve at least partial energy independence [9]. The development of the biofuel market promotes socio-economic development, supports employment growth in the fuel and energy sector, and influences the decarbonisation of the global economy in the long term [10]. It is therefore necessary to search for new, alternative, and competitive solutions, the deployment of which is supported by an economic balance sheet that takes into account the social and environmental impacts achieved.

1.2. The Potential of Microalgae

Biofuels produced on the basis of microalgal biomass have shown enormous potential for realisation [11]. Numerous scientific papers and large-scale plants have described in detail the parameters and technological systems of biofuel production using these microorganisms [12]. Reliable energy and environmental balances have also been drawn up for these solutions, making it possible to conduct a comprehensive life cycle analysis (LCA) for the individual technologies [13,14]. Microalgal biomass is a feedstock that can effectively compete with typical land crops, such as rapeseed, soya, or oil palm, in terms of the efficiency of the biosynthesis of lipids and their subsequent utilisation for biodiesel production [15]. In addition, biocomponents derived from microalgal biomass are treated as third-generation biofuels, which brings many benefits under the environmental policies of many countries, including those of the European Union (EU) [16].

Compared to terrestrial plants, microalgae are very tolerant, eurybiontic organisms that easily adapt to difficult or variable environmental conditions [17]. They do not require fertile arable soil for their growth and can be cultivated with wastewater and waste gases of various properties [18]. Intensive production systems for photosynthetic microalgal species are currently perceived as plants in which the process of CO₂ biostorage is efficient [19]. The advantage of energy systems based on the use of microalgal biomass is the fact that no herbicides or pesticides are used in their cultivation, which significantly reduces production costs and eliminates the possibility of secondary pollution of the natural environment [20]. Despite the undeniable strengths and benefits, there are also many problems associated with the operation of microalgae production plants and solutions that convert this type of biomass into energy sources. These include the overgrowth of biofilm on the walls of the photobioreactors, the self-shading of the cells, the high concentration of oxygen in the culture, the accumulation of compounds that are toxic to microalgal cells, the cost of nutrients, and the lighting of the photobioreactors (PBR) [21].

1.3. Heterotrophic Microalgae

The search for solutions to reduce the operational difficulties mentioned above and the need to reduce the cost of systems for cultivating photoautotrophic microalgal species has focused the work of many researchers on heterotrophic or mixotrophic cultures. It has been shown that some microalgal species are able to grow heterotrophically using organic carbon sources [22]. Heterotrophic cultures are characterised by higher biomass growth dynamics and much higher final concentrations of cells and lipids compared to phototrophic cultures [23]. In other experiments, microalgae have utilised many types of organic compounds, including fatty acids, technical glycerol, and most sugars [24]. The search is currently on for ways to use and analyse cheaper sources of organic media in the production process of microalgae, which are characterised by a high lipid content in the biomass, for the production of biodiesel. It has been shown that the use of waste glycerol in the heterotrophic microalgae cultivation process can not only lower operating costs but also reduce greenhouse gas emissions [25]. Due to the high production of this waste raw material, it is a burden on the environment, which is why new effective methods for its neutralisation are being sought. Technologies that utilise this organic substrate in the heterotrophic production of microalgae to produce biocomponents for fuels fit this trend [26]. The bioconversion of waste glycerol into high-value compounds is an important alternative to its purification [27]. It has been demonstrated that heterotrophic microalgae of the genus Schizochytrium sp. are able to metabolise waste glycerol and convert it into lipid storage substances that accumulate in the cells [28,29]. Global biodiesel production amounts to about 47.1 billion litres and is a process that co-produces waste glycerol, which accounts for more than 12% of the total amount of ester produced, regardless of the catalyst or technological process used [30]. Waste glycerol is also produced during bioethanol production and is estimated to account for an average of 10% of the total amount of sugar consumed [31]. Previous studies have shown that up to several hundred kilogrammes of lipids can be obtained from one tonne of crude glycerol during the heterotrophic cultivation of Schizochytrium sp. [32,33]. However, the final efficiency of this biological conversion depends on many factors related to both the properties of the waste glycerol and the technological parameters of the cultivation process [34,35].

1.4. Biodiesel from the Biomass of Schizochytrium sp.

It should be emphasised that work to determine the possibility of bio-oil production by heterotrophic microalgae of the genus Schizochytrium sp. has been carried out and described by many authors [32,33,34]. An effective optimisation of the process aimed at both intensifying the growth of the biomass of Schizochytrium sp. and increasing the level of synthesis of the lipid fraction has also been presented [36]. Depending on the source of the organic medium used, the initial substrate concentration in the culture medium, the method and frequency of dosing, the type of culture (batch, continuous, semi-continuous), and the length of a single culture cycle should be selected individually. It has been found that the optimum temperature is in the range of 26–27 °C, the nitrogen concentration is between 2 and 10 g/L, the pH of the culture is 7–8, and the oxygen concentration is not below 30% of the saturation level [29,34,35,36]. Published work also shows the possibility of using waste glycerol as a basic source of organic carbon for heterotrophic cultivation to produce biocomponents for fuels, including the quantity and quality of the lipid fraction produced. At the same time, there is a lack of studies documenting the use of biodiesel derived from the transesterification of bio-oil from heterotrophic Schizochytrium sp. culture to power internal combustion engines and the characteristics of exhaust emissions to the atmosphere. This was the basis and inspiration for this research topic.

This question is of great practical importance because it has been proven that the use of biofuels with a high content of fatty acid methyl esters (FAMEs) can lead to problems during transport and storage as well as in the supply of compression-ignition engines [37]. The main cause of the problems is the occurrence of deposits in the fuel system of the drive unit and the formation of deposits in the engine itself [38]. It has also been proven that the presence of trace substances in biofuels, such as free glycosides of sterols or saturated monoacylglycerols [39], has negative effects. For this reason, the World Wide Fuel Charter (WWFC) allows a maximum of 5% V/V FAMEs in diesel fuels of categories 1 to 5, provided that the fatty acid methyl esters fulfil the requirements specified in EN14214, ASTM D 671, or other equivalent standards [40]. A higher FAME content leads to many technical problems in the use of fuels, which results from their specific properties. Biodiesel has lower thermo-oxidative stability, which leads to rapid oxidation of the fuel mixture; has poorer flow properties at low temperatures, which leads to increased viscosity and clogging of the fuel filters; has high hygroscopicity, which causes emulsification and waterlogging, subsequently favouring microbiological contamination, corrosion, and coking of spray nozzles, which necessitates the use of detergent additives; and it tends to dissolve paint coatings protecting vehicle exteriors [41]. For this reason, European fuel manufacturers have set the maximum ratio of FAME to diesel oil at 7% (V/V) for technical reasons [42]. The characteristics of the required parameters and their permissible values were adopted in European standard EN590:2013-12 “Automotive Fuel-Diesel-Requirements and Test Methods” [43].

The main objective of the study was to present the emission characteristics of a compression-ignition engine fuelled with a mixture of diesel oil and biodiesel produced from lipids accumulated in the biomass of a heterotrophic culture of Schizochytrium sp. In addition, the efficiency of biomass and bio-oil production by Schizochytrium sp. based on waste glycerol was tested and the properties of the lipid fraction and biodiesel are presented.

2. Materials and Methods

2.1. Organisation of the Experiment

The experiments were conducted in three separate research stages. In stage 1, the microalgae Schizochytrium sp. were cultivated on a semi-industrial scale and the lipids accumulated in the biomass were analysed quantitatively. The culture conditions were established during previous optimisation work [30,44]. In stage 2, the lipid fraction was extracted from the biomass of Schizochytrium sp. and, subsequently, biodiesel was produced in the transesterification process. A qualitative analysis of the bio-oil and biodiesel was carried out. In stage 3, experiments were conducted to determine the emissions from a compression–ignition engine fuelled with a mixture of diesel oil and the biodiesel produced. In accordance with the applicable standards, the proportion of biodiesel in the fuel mixture was 7% V/V. Stage 3 was divided into five variants (V), the separation criterion of which was the tested engine load (EL), which were V1—EL0%, V2—EL25%, V3—EL50%, V4—EL75%, and V5—EL100% of the total power.

2.2. Materials

2.2.1. Origin and Cultivation of Schizochytrium sp. Biomass

The strain of Schizochytrium sp. from the ATCC (American Type Culture Collection) was used for the study. The method for the initial propagation of the biomass using the ATCC790By + culture medium was presented in our previous works [29,30,44].

2.2.2. Glycerol

The waste glycerol was sourced from the PKN Orlen Południe SA plant in Trzebinia, Poland. This raw material was used as the sole carbon source for the heterotrophic cultivation of Schizochytrium sp. The properties and characteristics of the waste glycerol are as follows: light brown colour, pH—5, glycerol—80% w/w, water—15% w/w, sulphated ash—5% w/w, methanol—0.3% w/w, non-glycerol organics—6% w/w, chlorides—10 ppm w/w, halogen derivatives—35 ppm w/w, acid content—0.25 mL/used NaOH, esters—8–10 mL/used NaOH, heavy metals—5 ppm w/w, aldehydes—10 ppm w/w, melting point—18 °C, boiling point—290 °C, flash point—177 °C, auto-ignition temperature—429 °C, decomposition temperature—>290 °C, vapour pressure—0.01 mbar, relative density at 20 °C—1.26 kg/L, and viscosity at 20 °C—1.5 mm²/second.

2.2.3. Diesel Oil

The diesel oil used, with the trade name VERVA ON type F, was manufactured by Orlen S.A., Poland. The properties of the product used are as follows: density at 15 °C—820.0–845.0 kg/m³, content of polycyclic aromatic hydrocarbons—max. 7.0% (m/m), sulphur content—max. 10.0 mg/kg, manganese content—max. 2.0 mg/L, flash point—min. 56.0 °C, water content—max. 200 mg/kg, content of solid impurities—max. 24 mg/kg, oxidative stability—max. 25 g/m³, min. 20 min, kinematic viscosity at 40 °C—2000–4500 mm²/s, FAME content—max. 7.0% (v/v), cold filter plugging point (CFPP)—max. −20 °C, and distillation residue—max. 2% (v/v).

2.3. Schizochytrium sp. Culture Conditions

The applied conditions and technological parameters of the heterotrophic cultivation of Schizochytrium sp. were selected on the basis of previous experimental and optimisation studies using the two-step Plackett–Burman identification method and the response surface method. The modelled parameter values and the final results were evaluated at small and semi-industrial scales under batch conditions as well as in semi-batch cultivation and continuous process [29,30].

A single cultivation cycle of Schizochytrium sp. lasted 120 h and the initial concentration of waste glycerol in the environment was 150 g/L. During the cultivation cycle, the actual concentration of glycerol in the running bioreactor was monitored cyclically (every 5 h). If the concentration was below 60 g/L, fresh waste glycerol was added in an amount that increased the concentration in the culture medium to 150 g/L. A glycerol concentration below 60 g/L reduced the cultivation efficiency due to the initiation of the stationary growth phase of Schizochytrium sp. [29,30]. The initial culture conditions were as follows: temperature 27 °C, peptone concentration 10 g/L, oxygen mass transfer rate kLa 600 1/h, salinity 17.5 psu, culture pH 6.5, yeast extract concentration 0.4 g/L, turbine speed 740 rpm, air flow rate 3.2 L/h, and initial inoculum concentration 5.0 g dry weight/L [38]. The cultivation of the heterotrophic microalgal biomass was carried out in a bioreactor with an active volume of 20 L (Biostat C, Sartorius Stedim, Aubagne, France). The bioreactor offered all operational possibilities in terms of aeration, mixing, ensuring a constant process temperature, sampling for analyses, media dosing, and pH control. The schematic and layout of the bioreactor are shown in Figure 1.

2.4. Biodiesel Production

The biomass obtained from the bioreactor was dried at 105 °C for 24 h. It was then ground in portions of 100 g using a mill (IKA Multidrive, IKA^®-Werke GmbH, Staufen, Germany) at 20,000 rpm for 60 s. The ground biomass was mixed with hexane. Hexane was added to the ground biomass at a dose of 300 mL/100 g of dried mass. Separation of the hexane from the biomass oil was carried out in a centrifuge (OHAUS Frontier 5000 Multi-Pro FC5917RF Short, Parsippany, NJ, USA) at 11,000 rpm for 3 min. After hexane evaporation, the oil was then transesterified. The transesterification reaction was carried out for 2 h in a glass reactor with complete stirring. The process was carried out at 65 °C and atmospheric pressure, maintaining a molar ratio of microalgae oil to methanol of 1:9. NaOH was used in an amount of 2% (w/w) of the oil mass as a homogeneous transesterification catalyst. The waste glycerol was then obtained from the biodiesel and rinsed with distilled water containing acid at a concentration of 5% in the next step. The biodiesel was separated using a rotary evaporator at 80 °C and then dried at 100 °C.

2.5. Compression–Ignition Engine

The experiments to determine the emissions were carried out with a vertical, water-cooled, single-cylinder diesel engine with compression ignition (Kirloskar AV1, Pune, India). The basic parameters characterising the engine used are as follows: rpm—1500, engine cooling—water, number of cylinders—1, bore x stroke—80 mm × 110 mm, cubic capacity—0.553 L, compression ratio—16.5:1, rated output—3.7/5 kW/Hp, torque at full load—2.387 kgm, fuel consumption—185 + 5% gm/hp-hr, lube oil capacity—3.7 L at higher level mark on dipstick, lube oil consumption—0.8% of SFC max., fuel tank capacity—6.5 L, dimension L × W × H—617 mm × 504 mm × 843 mm, and engine weight—130 kg.

2.6. Analytical Methods

The dry cell weight (DCW) of Schizochytrium sp. was determined by drying and weighing the pre-centrifuged (50 mL biomass, 8000× g for 15 min, UNIVERSAL 320 R centrifuge, Hettich, Tuttlingen, Germany) and distilled water-washed biomass sample at 60 °C for 12 h in a moisture analyser (MAR, Radwag, Radom, Poland). The protocol corresponded to the method described by Chang et al. (2013) [45]. The concentration of glycerol in the bioreactor was detected by centrifugation (8000× g, 4 min, 10 °C; UNIVERSAL 320 R centrifuge, Hettich, Tuttlingen, Germany) and then analysing the content of this compound in the supernatant (Glycerol GK Assay Kit, Megazyme, Bray, Ireland). The fat concentration in the freeze-dried biomass was determined using hydrochloric acid (ALPHA 1–4 LD plus freeze dryer, Martin Christ Gefriertrocknungsanlagen GmbH, Hamburg, Germany). The prepared biomass was placed in a water bath (temp. 75 °C, 40 min, GFL 1003, GFL, Burgwedel, Germany). The microalgal biomass was mixed with n-hexane and placed in a vacuum evaporator (Hei-VAP Advantage G3, Heidolph, Schwabach, Germany) and then the lipid content was measured gravimetrically. The fatty acids were determined using the direct transmethylation method. The phase with FAMEs was collected and subjected to chromatographic analysis (Clarus 680 GC gas chromatograph (Perkin Elmer, Waltham, MA, USA).

The characteristics of the oils were determined according to the standard methods listed below: acid value—EN 14104 [46], calorific value—DIN 51900 [47], cetane number—EN ISO 5165 [48], carbon residue—EN ISO 10370 [49], density at 15 °C—ISO 3675 [50], flash point—ISO 15267 [51], phosphorus content—ISO 10540 [52], iodine value—EN 14111 [53], oxidative stability 110 °C—EN 14112 [54], sulphur content—ISO 3987 [55], total contamination—EN 12662 [56], water content—EN ISO 12937 [57], and viscosity at 40 °C—ISO 3104 [58].

The degree of conversion of the triglycerides in the transesterification process was determined using high-performance liquid chromatography HPLC (C-18 column, two DAD detectors with wavelength λ = 205 nm, flow rate 0.9 cm²/min, column temperature 25 °C, injection volume 1.0 µL) (LC-10AT, Shimadzu, Kyoto, Japan). A mixture of solvent A—isopropanol-hexane (4/5) —and solvent B—methanol—was used as the mobile phase according to the following gradient: 0 min—solvent A 100%, 20 min—solvent A 100%, 45 min—solvent B 100%, 70 min—solvent B 100%, 71 min—solvent A 100%, and 75 min—solvent A 100%.

The transesterification products were analysed using a GC (10:1—split ratio, 100 °C—initial column temperature, 0.80 mL/min—column flow rate, 40 mL/min—make-up gas flow rate, temperature gradient: 4.0 °C/min—185 °C, 0.5 °C/min—220 °C, 5.0 °C/min—240 °C) (GC-15A Shimadzu, Kyoto, Japan) with an Rt-2560 column (110 m × 0.20 µm ID, film thickness 0.25 mm). The analyses were carried out using the following analytical standards: Food Industry FAME Mix (C4:0–C24:1) and FAME Mix (C18:0–C20:0). The exhaust gas composition was analysed using an Infralyt N-V101 analyser (Test-Therm Ltd., Krakow, Poland). CO, CO₂, and HC (hydrocarbons) were measured optically with an infrared beam, NOX—with an electrochemical method. Measuring ranges: CO (0–2000 ppm), CO₂ (0–20%), HC (0–2500 ppm), NO (0–2500 ppm), and NO₂ (0–500 ppm). Operating conditions: ambient pressure 860–1060 hPa, mains voltage AC 230 V ± 10% (50 Hz ± 2%), sample gas temperature at the probe tip 5–500 °C, operating temperature 5–40 °C, and power consumption max. 60 VA. The exhaust gas opacity was measured using an AVL 439 opacimeter (AVL List GmbH, Graz, Austria).

2.7. Statistical Methods

STATISTICA 13.3 PL software (Statsoft, Inc., Tulsa, OK, USA) was used for the statistical analyses. Testing of the hypothesis regarding the distribution of the analysed variables was based on the Shapiro–Wilk W-test. One-way analysis of variance and Levene’s test were used to analyse the significance of differences between and within groups. Tukey’s HSD test was used to test the significance between the analysed variables. The significance level assumed in the tests was α = 0.05.

3. Results and Discussion

3.1. Schizochytrium sp. Biomass Growth and Lipid Production

In stage 1, a heterotrophic culture of Schizochytrium sp. biomass was established, which served as a source for the bio-oil tested in the subsequent stages of the study. During the culture, three phases of microalgal population growth could be distinguished. The adaptation phase (lag phase) lasted from 0 to 40 h, the logarithmic growth phase was observed between 40 and 80–100 h of operation of the bioreactor, and the stationary growth phase was observed from 80–100 to 120 h of incubation. In the lag phase, the biomass concentration increased from the initial value of 5.2 ± 1.3 g/L to 43.10 ± 5.2 g/L, and in the logarithmic growth phase, the DCW value reached a concentration of 133.9 ± 10.7 g/L (Figure 2). In the following hours of cultivation, an increase in the concentration of Schizochytrium sp. biomass to 140.7 ± 13.9 g/L was observed, although the observed differences were not statistically significant (Figure 2). The biomass growth rate in the logarithmic growth phase was 1.34 ± 0.2 g/L·h.

To ensure a high growth rate of the Schizochytrium sp. population, many factors must be ensured and controlled. Among the most important are the type of organic substrate, the type of dosage, the intensity of mixing, the oxygen concentration in the environment, and the type of cultivation [59]. Previous studies have shown that the highest efficiency of biomass production of Schizochytrium sp. can be achieved in a batch culture [60], where 200 g dry mass/L was reached. However, it should be emphasised that, in this case, the cultivation was carried out at laboratory scale and the carbon source was very easily digestible and metabolised glucose. By contrast, Chi et al. [61] achieved a significantly lower biomass production of 22.1 g/L for Schizochytrium limacinum strain SR21 when glycerol was used as the carbon source. It was found that a two-stage culture was the most efficient, divided into a first stage to increase cell number and a second stage to increase cell size by lipid accumulation. In this protocol, the concentration of microalgal biomass in the bioreactor was 37.9 g/L. In the same study, the optimal range for the concentration of crude glycerol was given as 75–100 g/L [61]. In our own studies, the investigations were conducted on a fractionated technical scale under operating conditions close to real conditions. Therefore, a continuous culture was used, which was carried out under conditions that had been optimised in the authors’ previous studies [29,44].

The lipid concentration in the culture correlated with the density of the Schizochytrium sp. population in the culture system. At the beginning of the culture, it was 4.4 ± 0.7 g/L (Figure 2). In the logarithmic growth phase, the concentration of lipid substances in the bioreactor increased to 57.0 ± 2.2 g/L after 100 h of culture (Figure 2). At the end of a single culture cycle, the lipid concentration was statistically comparable and totalled 58.2 ± 1.1 g/L (Figure 2). The changes in glycerol concentration in the culture medium varied between 150 ± 8.2 g/L and 55.6 ± 5.2 g/L after 20 h of culture (Figure 2). In accordance with the operational assumptions of the experiment established during production optimisation [29,30], this medium component was systematically supplemented after reaching a level close to 60 g/L. Preliminary optimisation studies [29] demonstrated that Schizochytrium sp. biomass cells accumulated 48.85 ± 0.81 g/L of lipids during a 120 h culture period. According to Ratledge [62], lipid accumulation is caused by a deficiency of a certain nutrient, usually nitrogen, in the culture medium. During the growth phase, when nitrogen sources are depleted, excess carbon in the medium continues to be taken up by the cells and converted into reserve lipids [62]. Previous studies have shown that the parameters that significantly influence the growth of microalgae and lipid accumulation in the biomass of Schizochytrium sp. are the temperature conditions and the glycerol content in the environment. Adequate oxygen levels and the presence of peptone are also indicated [30]. Temperatures above 26 °C have been shown to stimulate the biomass growth dynamics of Schizochytrium sp. [30]. This was confirmed in the study by Wen and Chen [63], which showed the relationship between microalgal biomass growth and lipid accumulation in water and ambient temperature conditions. Liu et al. [64] showed that the availability of intracellular oxygen increases at low temperatures. This phenomenon activates oxygen-dependent enzymes that catalyse the desaturation and elongation of PUFAs in the biomass. The increase in lipid content in microalgal cells at low temperatures is associated with an increase in the elasticity of the cell membrane [65].

3.2. Bio-Oil Characteristics and Properties

All determined basic parameters of the recovered bio-oil, which are listed in

Table 1. Basic properties of the tested bio-oils and the fatty acid composition in the lipid fraction produced by Schizochytrium sp.

Parameter	Unit	Value	EN 14214 Standard
Density at 15 °C	kg/m3	873 ± 39	860–900
Viscosity at 40 °C	mm2/s	4.3 ± 1.3	3.5–5.0
Flash point	°C	139 ± 4	>101
Total contamination	mg/kg	6.7 ± 0.5	<24
Oxidative stability, 110 °C	hours	8.9 ± 0.5	>8
Acid value	mgKOH/g	0.38 ± 0.11	<0.5
Iodine value	gI/100 g	98.4 ± 3.8	<120
Cetane number	-	55.2 ± 0.3	>51
Water content	mg/kg	82.1 ± 4.3	<500
Sulphur content	mg/kg	3.9 ± 0.8	<10
Phosphorus content	mg/kg	2.4 ± 0.9	<4
Fatty acid	Unit	Value	Numerical symbol
Myristic acid	% Total Fatty Acids	3.1 ± 0.4	C14:0
Palmitic acid		40.4 ± 2.8	C16:0
Stearic acid		4.6 ± 1.1	C18:0
Eicosapentaenoic acid		6.7 ± 0.9	C22:5
Docosahexaenoic acid		43.8 ± 2.3	C22:6

, fulfilled the conditions of the standard EN 14214 published by the European Committee for Standardisation, which describes the requirements for bio-components of fuels, FAMEs, and biodiesel [66]. The density and viscosity of the recovered bio-oil were 873 ± 39 kg/m³ and 4.3 ± 1.3 mm²/s, respectively, which were within the average range of the EN14214 standard, which covers values from 860 to 900 kg/m³ and 3.5–5.0 mm²/s, respectively. The flash point was 139 ± 4 °C, well above the standardised minimum value of 101 °C. The setting of the flash point value in the EN 14214 standard above 101 °C is determined by many aspects related to the operational and utilisation activities in the biofuel sector. This is mainly due to the fact that biofuel is less volatile and therefore less likely to ignite under normal conditions. This reduces the risk of accidental spontaneous combustion during storage, transport, and maintenance at refuelling stations [67]. A higher flash point contributes to more stable and even combustion in the engine. Fuel with a lower flash point can lead to problems with the stable composition of the fuel mixture and uneven combustion, as it tends to vaporise quickly [68]. In general, a higher flash point reduces the risk of toxic smoke emissions and improves the stability of the fuel, which is important for operator safety and the quality of emissions to the environment [69].

An important parameter for characterising substrates for biodiesel production is the acid number. It is assumed that a low value of this parameter is advantageous for internal combustion engines, as it reduces the risk of corrosion and improves the oxidative stability of the biofuel [70]. It has also been shown to have a direct effect on the efficiency of biodiesel production, as it limits the formation of soaps, which, in turn, make it difficult to separate the biodiesel from glycerol and other by-products [71]. In the in-house studies presented, the acid number of the analysed bio-oil was within the norm (<0.50 mgKOH/g), but in several cases, it was close to the upper limit. The average value was 0.38 ± 0.11 mgKOH/g. The iodine value was significantly lower than the maximum value specified in the EN 14214 standard (<120 gI/100 g) and was 98.4 ± 3.8 gI/100 g. A low iodine value, similar to the acid value, increases the oxidative stability of the fuel but also improves the operating parameters by reducing the risk of deposits and engine corrosion [72]. A low value of this parameter improves the fluidity of biofuels at low temperatures, optimises the combustion process, and helps to meet the quality standards for the emitted exhaust gas. It has been proven that biofuels with a low iodine value are more durable, more energy efficient, and less problematic when used in diesel engines [73]. A high iodine value can bring certain advantages, such as improved lubricating properties and better solubility of the biofuel. However, these benefits are minor and do not compensate for the challenges mainly associated with the operational properties of biofuels [74].

The bio-oil tested in the experiments was characterised by a low total contamination of 6.7 mg/kg. A high level of contamination leads to problems during transesterification, can cause damage to the equipment, and also reduces the stability of the fuel [75]. The requirements of the EN 14214 standard assume that the concentration of impurities does not exceed 24 mg/kg. The sulphur and phosphorus contents were 3.9 ± 0.8 mg/kg and 2.4 ± 0.9 mg/kg, respectively, and therefore complied with the standard. The correct values of these indicators are important for the operational properties of biofuels in terms of reducing deposits, clogging of the plant, its corrosion, and also environmental aspects, in particular the reduction of acid gas emissions [76].

The qualitative composition of fatty acids in the lipid fraction of the biomass of Schizochytrium sp. was dominated by docosahexaenoic acid (DHA). Its content was 43.8 ± 2.8%. This is a polyunsaturated fatty acid that is mainly found in fish fat and some vegetable oils such as linseed oil or microalgae oil [77]. In the context of biodiesel production, DHA has a significant disadvantage related to the presence of many double bonds [78]. This has a direct impact on the high iodine value, which indicates lower oxidative stability and deterioration of fuel properties over time, e.g., during transport and storage [79]. A high DHA content can lead to faster oxidation, which may require additional stabilisation and control measures to ensure the quality of biodiesel. Oxidation can lead to the formation of acids, residual polymeric compounds, and other products that can reduce the quality of biodiesel and affect its stability [80]. In practice, DHA is often present in some vegetable and microalgae oils, but its presence requires careful management to ensure optimal properties and stability of biofuels. Usually, the simplest and most practical technological procedure is to blend substrates with high DHA content with other lipid substrates [81].

Palmitic acid (C 16:0), characterised by a comparable content in bio-oil, was obtained from the biomass of Schizochytrium sp. and plays a very important role in the biorefinery industry due to its properties [82]. The content of this FAME component in the lipid profile was 40.4 ± 2.8%. Palmitic acid, as a saturated fatty acid, is less susceptible to oxidation compared to polyunsaturated fatty acids [83]. Therefore, biodiesel with a higher palmitic acid content has better oxidative stability, which has a direct impact on the extension of its storage time and a lower risk of degradation [84]. Palmitic acid influences the viscosity of biodiesel and the pour point. A high palmitic acid content can lead to a higher viscosity and pour point of biodiesel, which can affect its poorer operational properties, especially under cold conditions [85]. In practice, controlling the palmitic acid content and blending with other oils can help to optimise the properties of biodiesel and ensure that it meets quality standards and performs adequately under different operating conditions [86].

A significantly lower concentration of 6.7 ± 0.9% was found for decosapentaenoic acid (DPA), the properties of which are similar to those of DHA. The contents of stearic acid (C 18:0) and myristic acid (C 14:0) were 4.6 ± 1.1% and 3.1 ± 0.4%, respectively. These are saturated fatty acids that can be effectively utilised in the production of biodiesel [87]. In studies by Kujawska et al. [29,30], the profile of fatty acids produced by the microalgae Schizochytrium sp. did not differ, regardless of the scale of the process. Saturated palmitic acid (C 16:0) and unsaturated docosahexaenoic acid (C 22:6) were the major fatty acids in the lipid fraction produced by the microalgae. The percentage composition of the fatty acids was the same for all test variants. The lowest concentration in the cells of Schizochytrium sp. biomass was observed for stearic acid (C 18:0) and ranged from 1.47 ± 0.13% of TFA in batch culture to 2.72 ± 0.43% of TFA in continuous culture [29,30]. Similar observations were made by Hu et al. [88], who showed that the quality profile of fatty acids with a chain length of 10–24 atoms was similar among species of the same class or group and did not depend on the scale of cultivation. Xu et al. [89] showed that the composition of fatty acids changed depending on the growth phase of the biomass of Pavlova viridis. An increase in their concentration was observed in the logarithmic growth phase and a stabilisation or slight decrease in the stationary growth phase [89]. Petel et al. [90] pointed out that the degree of unsaturation of the fatty acids produced by microalgae can be altered by various parameters, such as temperature, concentration of carbon and nitrogen compounds, carbon source, degree of dilution in the case of continuous culture, and oxygen saturation.

3.3. Engine Emissions

In accordance with the applicable standards, the proportion of biodiesel in the fuel mixture was 7% V/V. It was found that the concentration of carbon dioxide (CO₂) in the exhaust gas increased with the tested engine load (EL). In V1 (0%EL), the CO₂ concentration was 2.7 ± 0.4% (Figure 3). A successive, significant increase in CO₂ emissions was observed in the subsequent variants V2–V4, where the CO₂ content rose from 3.9 ± 0.9% (25%EL) to 6.1 ± 1.7% (75%EL) (Figure 3). The full engine load in V5 had no statistically significant influence on the increase in CO₂ emissions, the proportion of which in the exhaust gas was 6.7 ± 1.2% (Figure 3). The relationship between the increase in CO₂ emissions in the exhaust gas and the engine load corresponded to a linear function. A strong positive fit of the model to the empirical data was found, which amounted to R² = 0.9749 (Figure 3).

Similar observations were made by Aydın and Ogüt [91] and Sanjid et al. [92], who linked them to higher fuel consumption in the range of higher engine loads. Similarly, the study by Namitha et al. [93] found higher CO₂ content in the exhaust gas at higher engine loads when burning biodiesel from Chlorella sorokiniana and Monoraphidium sp. compared to pure diesel fuel. Kilic et al. [94], on the other hand, made different observations and showed that the use of blends of bio-oil and diesel fuel or biodiesel led to a reduction in the CO₂ concentration in the exhaust gas. Abed et al. [95] showed lower CO₂ emissions with biodiesel blends from jatropha, oil palm, and algae compared to diesel fuel and explained this with a higher oxygen content in biodiesel blends. Bazooyar et al. [96], who determined the combustion efficiency and emissions of an industrial boiler with biodiesel and diesel fuel, also found lower CO₂ emissions for biodiesel. For comparison purposes,

Table 2. Comparison of the CO₂ concentration in the exhaust gas as a function of the fuel used and the engine load level.

Fuel	Engine Load [%]	CO2 [%]	Ref.
Diesel and biodiesel blend from Schizochytrium sp. lipids	0	2.7 ± 0.4	current results
	25	3.9 ± 0.9
	50	5.3 ± 1.3
	75	6.1 ± 1.7
	100	6.7 ± 1.2
Diesel	0	2.1 ± 0.5	[97]
	25	2.9 ± 0.3
	50	4.1 ± 0.2
	75	5.2 ± 0.3
	100	5.9 ± 0.2
Rapeseed oil biodiesel	0	2.4 ± 0.1
	25	3.3 ± 0.2
	50	4.9 ± 0.4
	75	5.7 ± 0.1
	100	6.3 ± 0.3

shows the CO₂ emission values of the same tested engine depending on the tested fuel. Regardless of whether only rapeseed oil [97], pure biodiesel from rapeseed oil [97] or a mixture of diesel and biodiesel from Schizochytrium sp. was used, a linear increase in CO₂ emissions was observed, which correlated with the tested engine load. At an engine load of 100, significantly higher emissions of this indicator were observed for biodiesel than for diesel fuel (Table 2).

A different pattern of changes in the concentrations of emitted gases, which correlated with the applied EL, was observed in the case of CO. For this indicator, the emissions decreased significantly with the increase in EL in the range from V2 (25%EL), where 830 ± 98 ppm was observed, to V5 (100%EL), where the CO concentration was 480 ± 31 ppm (Figure 4). In V1 (0%EL), a lower average concentration of this exhaust component was observed, at the level of 850 ± 70 ppm, but this value did not differ significantly from that in V2 (25%EL) (Figure 4). The linear function used in the analysis of CO emissions showed a strong negative relationship between the EL level and the observed concentration of this indicator. The value of the coefficient of determination R² for the entire range of output data analysed was 0.9428 (Figure 4).

According to Gad et al. [98], CO emissions are mainly caused by an excessively rich fuel-air mixture that forms in the engine and a low combustion temperature. It has been proven that higher CO emissions occur at low load. This is due to the higher density and viscosity of biodiesel and the lean mixture, the low temperature in the cylinder, and the poor atomisation of this type of fuel [99]. On the other hand, a decrease in CO emissions is observed with biodiesel and diesel fuel at higher loads (

Table 3. Comparison of the CO concentration in the exhaust gas as a function of the fuel used and the engine load level.

Fuel	Engine Load [%]	CO [ppm]	Ref.
Diesel and biodiesel blend from Schizochytrium sp. lipids	0	850 ± 70	current results
	25	830 ± 98
	50	640 ± 112
	75	540 ± 46
	100	490 ± 31
Diesel	0	700 ± 78	[97]
	25	840 ± 46
	50	570 ± 38
	75	490 ± 25
	100	360 ± 41
Rapeseed oil biodiesel	0	1040 ± 82
	25	1200 ± 77
	50	930 ± 50
	75	670 ± 29
	100	510 ± 25

). This is due to the increase in cylinder temperature and the improvement in molecular oxygen concentration, which, in turn, promotes oxidation and reduces viscosity [100]. Perumal et al. [101] showed that an increase in engine load causes a decrease in the air demand coefficient. This leads to more efficient combustion and lower CO emissions [101]. The results of the studies by Arunkumar et al. [102] and Rajak et al. [103] also confirmed the above observations. However, the study by Azad et al. [104] found significantly lower CO emissions when burning biodiesel from macadamia and grape seeds compared to diesel fuel. This was explained by the more efficient oxidation of carbon caused by a higher oxygen content in biodiesel [104]. When using a mixture of biodiesel from microalgae, significantly higher CO emissions were observed compared to diesel fuel. At the same time, lower concentrations were observed than when the engine was fuelled only with biodiesel from rapeseed oil (Table 3). In this case, the CO concentration in the exhaust gas ranged from 510 ± 25 ppm to 1200 ± 77 ppm, depending on the engine load used [97]. In the variant in which only conventional fuel was tested, values of 360 ± 41 to 840 ± 46 ppm were achieved (Table 3).

The lowest NOx concentration in the exhaust gas was observed at the lowest tested EL in V1. In this variant, it was 91 ± 12 ppm and increased with increasing EL level (Figure 5). The most dynamic increase in the emissions of this gas was observed in V2 (25%EL) and V3 (50%EL), where the NOx concentrations were 166 ± 21 ppm and 319 ± 19 ppm, respectively (Figure 5). In V4 (75%EL) and V5 (100%EL), the increase in emissions was not statistically significant and the NOx levels were 370 ± 33 ppm (V4) and 397 ± 37 ppm (V5), respectively (Figure 5). A positive linear correlation was observed between the analysed variables. It was found that NOx emissions increased proportionally with the increase in engine load. The coefficient of determination was R² = 0.9329 (Figure 5).

The NO_x content in the exhaust gas produced by biodiesel compared to diesel fuel is still unclear, as researchers have obtained contradictory conclusions [105]. In the works of Miri et al. [106] and Özener et al. [107], it was shown that the use of biofuels caused an increase in NO_x emissions. It has been shown that NO_x emissions increase with the applied excess air ratio and with temperature [108]. However, they can be minimised through the use of exhaust gas recirculation (EGR) or other additives [109]. Contrasting research results were presented by Wahlen et al. (2013) [110], among others, showing that heat release was high in biodiesel from microalgae, while NO_x emissions were reduced by about 14% compared to diesel fuel. Chen et al. [111] explained this by the short combustion time, which provides a limited time for the conversion of N₂ to NO_x, lowers the average gas temperature, and leads to a reduction in NO_x emissions. The authors’ previous studies showed that the NO_x content in the exhaust gas was comparable and independent of the type of fuel used. The use of diesel oil, pure biodiesel from rapeseed oil [97], or the fuel mixture tested in the present study had no significant influence on the changes in NO_x emissions. The decisive factor was the tested engine load (

Table 4. Comparison of the NO_x concentration in the exhaust gas as a function of the fuel used and the engine load level.

Fuel	Engine Load [%]	NOx [ppm]	Ref.
Diesel and biodiesel blend from Schizochytrium sp. lipids	0	91 ± 12	current results
	25	166 ± 21
	50	319 ± 19
	75	370 ± 33
	100	397 ± 37
Diesel	0	111 ± 21	[97]
	25	169 ± 18
	50	332 ± 32
	75	390 ± 15
	100	457 ± 31
Rapeseed oil biodiesel	0	96 ± 17
	25	147 ± 31
	50	272 ± 9
	75	301 ± 20
	100	376 ± 32

).

The HC emissions increased significantly from 33 ± 2 ppm to 39 ± 5 ppm in the range of the tested EL from 0% (V1) to 50% (V3) (Figure 6). In the next tested experimental variants, namely V4 (EL75%) and V5 (EL100%), the HC concentration in the emitted gases remained at a constant level of 38 ± 3 ppm and 38 ± 7 ppm (Figure 6). No close correlation was found between the increase in HC emissions in the exhaust gas and the engine load. The coefficient of determination in this case was R² = 0.6712 (Figure 6). Significantly lower HC emissions were observed when the drive unit was operated only with biodiesel produced on the basis of rapeseed oil [97]. In this case, the HC concentration in the exhaust gas was between 27 ± 2 ppm and 35 ± 6 ppm, depending on the engine load tested (

Table 5. Comparison of the HC concentration in the exhaust gas as a function of the fuel used and the engine load level.

Fuel	Engine Load [%]	HC [ppm]	Ref.
Diesel and biodiesel blend from Schizochytrium sp. lipids	0	33 ± 2	current results
	25	34 ± 3
	50	39 ± 5
	75	38 ± 3
	100	38 ± 7
Diesel	0	44 ± 7	[97]
	25	46 ± 6
	50	40 ± 4
	75	42 ± 8
	100	42 ± 2
Rapeseed oil biodiesel	0	27 ± 2
	25	32 ± 1
	50	35 ± 6
	75	33 ± 2
	100	30 ± 5

). With diesel fuel, the values ranged from 40 ± 4 ppm to 46 ± 6 ppm [97].

HC emissions are caused by incomplete combustion of the fuel in the engine [112]. According to Zahos-Siagos et al. [113], this phenomenon can be due to too low oxygen concentration in the chamber, too low combustion temperature, and too short residence time of the fuel at the ignition temperature. Han et al. [114] emphasised that the properties of biodiesel, in particular the higher oxygen content in the fuel, indicate that HC emissions should be lower compared to diesel fuel. This was confirmed by the results of our own research and the studies presented by Gharehghani et al. [100] and Palash et al. [115]. However, this theory was challenged by the research results described by Nabi et al. [116], who observed a maximum 13% higher HC emissions in the case of the Licella biofuel blend compared to diesel fuel. This could be due to the higher density of biodiesel, which has a direct effect on the increase in the size of the atomised fuel droplets. This, in turn, can lead to incomplete combustion of the fuel and consequently to increased HC emissions [117]. This phenomenon is less significant at higher engine loads, as a richer fuel-air mixture is then injected into the combustion chamber and the air–fuel ratio is therefore higher. As a result, combustion is more efficient and HC emissions are lower [118].

The lowest, statistically comparable smoke opacity values of the exhaust gas were observed in V1 (0%EL) and V2 (25%EL). The measured values were 13 ± 2% and 15 ± 2%, respectively (Figure 6). Testing higher engine load led to a significant increase in this emission factor. In V3 (EL50%), the smoke opacity reached an average value of 23 ± 4%, while in V4 (EL75%), it was 36 ± 6% (Figure 7). By far, the highest emissions of this type of pollutant were observed in V5, where the applied load of the energy unit was 100%. In this case, the smoke opacity of the emitted gases was 53 ± 8% (Figure 7). The linear function showed a positive correlation between EL and smoke opacity, which was described by the coefficient of determination at the level of R² = 0.9207 (Figure 7).

Biodiesel from microalgae is partially aerated, which promotes the oxidation of combustible particles [119]. Biodiesel also has a lower calorific value than diesel fuel, which enables a lower combustion temperature and lower combustion pressure, thus contributing to lower smoke emissions [120]. Previous work by the authors showed that the direct influence of the fuel used on the observed smoke opacity was significant at higher EL levels [97]. When the engine was fuelled with heating oil only, the values of this pollution indicator ranged between 31 ± 3% (EL50%) to 66 ± 10% (EL100%), while with biodiesel from rapeseed oil, they ranged between 25 ± 3% (EL50%) to 59 ± 3% (EL100%) (

Table 6. Comparison of smoke opacity as a function of the fuel used and the engine load level.

Fuel	Engine Load [%]	Smoke [%]	Ref.
Diesel and biodiesel blend from Schizochytrium sp. lipids	0	13 ± 2	current results
	25	15 ± 2
	50	23 ± 4
	75	36 ± 6
	100	53 ± 8
Diesel	0	3 ± 1	[78]
	25	12 ± 4
	50	31 ± 3
	75	44 ± 6
	100	66 ± 10
Rapeseed oil biodiesel	0	9 ± 2
	25	13 ± 1
	50	25 ± 3
	75	36 ± 5
	100	59 ± 3

).

4. Conclusions

The heterotrophic cultivation of Schizochytrium sp. based on waste glycerol allowed the final DCW concentration to be increased to 140.7 ± 13.9 g/L in a single cultivation cycle. The biomass growth rate in the logarithmic growth phase was 1.34 ± 0.2 g/L·h. The lipid concentration achieved in the technological system was 58.2 ± 1.1 g/L.

The qualitative composition of fatty acids in the lipid fraction of Schizochytrium sp. biomass was dominated by decosahexaenoic acid and palmitic acid, the contents of which were 43.8 ± 2.8% and 40.4 ± 2.8%, respectively. All of the basic parameters determined for the bio-oil obtained fulfilled the requirements of the EN 14214 standard published by the European Committee for Standardisation, which describes the requirements for bio-components of fuels.

In most cases, with the exception of CO and HC, the observed pollutant concentrations in the exhaust gas of a compression-ignition engine increased linearly with the increase in the applied load of the drive unit. No significant technical or operational problems were observed when the engine was fueled with the tested fuel mixture.

At 100% engine load, higher CO₂ emissions were observed with the biodiesel blend compared to diesel oil. For the mixture of crude oil and biodiesel from the lipids of Schizochytrium sp., higher CO emissions were observed over the entire load range of the engine. The NO_x content in the exhaust gas was comparable and independent of the type of fuel used. The engine load had a direct influence on the observed hydrocarbon emissions and smoke opacity when biodiesel was used. However, lower hydrocarbon emissions were observed for biodiesel compared to crude oil at lower engine load values. An inverse relationship was documented for smoke opacity.

The direction of further research should concern two main aspects. The first should continue to focus on the technological and economic optimisation of lipid accumulation in microalgal biomass and subsequent biodiesel production. Research under realistic conditions carried out on pilot, semi-industrial, and technical scales is particularly important. The second significant area of research concerns the development of engine design solutions that enable the safe and efficient use of fuels with a higher proportion of bio-components, which will have a direct impact on further reducing the consumption of fossil fuels.