
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12374-2
10.1016/j.heliyon.2024.e36343
e36343
Research Article
The potential of third-generation biodiesel from Tolypothrix sp. CACIAM22 as a feedstock
Terra de Oliveira Deborah deborahterra.o@hotmail.com
ab⁎
de Jesus Paiva Rutiléia ab
Albuquerque de Mescouto Vanessa ab
Ferreira da Silva Sara Roberta a
Farias Da Costa Ana Alice a
Santos Agenor Valadares b
Gonçalves Evonnildo Costa b
Narciso da Rocha Filho Geraldo a
Rodrigues Noronha Renata Coelho c
Santos do Nascimento Luís Adriano adriansantos@ufpa.br
ab⁎⁎
a Amazon Oil Laboratory, Guamá Science and Technology Park, Belém, 66075-750, Brazil
b Graduation Program of Biotechnology, Institute of Biological Sciences, Federal University of Pará, Belém, 66075-110, Brazil
c Laboratory of Genetics and Cell Biology, Center for Advanced Studies of Biodiversity, Institute of Biological Sciences, Federal University of Pará, Augusto Corrêa Street, Guamá, Belém, 66075-110, PA, Brazil
⁎ Corresponding author. deborahterra.o@hotmail.com
⁎⁎ Corresponding author. adriansantos@ufpa.br
19 8 2024
30 8 2024
19 8 2024
10 16 e363438 4 2024
13 8 2024
13 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Renewable energy has been recognized as an alternative to fossil fuels as a step to transform the energy produced and consumed worldwide. Cyanobacteria and microalgae are currently being considered as substitutes to the traditional feedstock used to produce biofuels due to their ability to achieve high amounts of lipids under cellular stress conditions. The aim of this study was to investigate the utilization of Tolypothrix sp. CACIAM 22 cyanobacterial biomass as a feedstock for biodiesel production, specifically by examining the effects of supplementing with hydrolysate of Brazil nutshell (HBNS) on biomass generation, lipid production, fatty acid composition, and quality of synthesized biodiesel. The supplementation of HBNS led to a significant increase of 12g.L−1 in wet biomass production. The lipid content reached 41 % of the biomass produced in HBNS supplemented cultures when nitrate source was deprived. The quality evaluation of cyanobacteria-derived biodiesel was performed using Biodiesel Analyzer ver 2.2 software, revealing superior quality compared to biodiesel produced from plant sources. The biodiesel exhibited values of 23 h for oxidative stability, 65 for cetane number, and an iodine index of 31 (g I2. 100 g−1fat), indicating promising potential as a renewable source. This study is the first to utilize HBNS as an organic supplement for cyanobacteria culture medium and assess its impact on biomass and lipid production in Tolypothrix sp., supporting the hypothesis of utilizing this biomass as a renewable feedstock for biodiesel production as a viable alternative to plant sources based on biomass production, lipid productivity, and biodiesel quality.

Graphical abstract

Image 1

Keywords

Cyanobacteria
Lignocellulose
Biodiesel
Brazil nutshell
Hydrolyzed
Amazon
==== Body
pmc1 Introduction

Biofuels are called biomass derived from sustainable sources such as plants, microorganisms and forest residues that have the potential to replace fossil fuels. The production of biofuels is classified into four generations, each differentiated by the type of raw material used and the technology used. When problems in obtaining raw materials or market difficulties arise, the next generation emerges to overcome these obstacles. Fig. 1 below provides an illustrative representation of these generations [[1], [2], [3]].Fig. 1 Biofuels generations and their raw materials.

Fig. 1

First-generation biofuels, which are still being produced today, utilize oilseed crops such as soybeans, palm, and canola for biodiesel production. Additionally, ethanol production involves the use of sugarcane in Brazil and corn in the United States, the two largest producers of ethanol globally [4].

The second generation of biofuels aims to reduce the reliance on food crops as raw materials, although the technologies associated with its production remain relatively costly. To produce a sustainable and clean biofuel from second-generation of biofuels has still been a challenge because it involves toxic solvents for pretreatments of the lignocellulosic biomass and separation of its layers and requires the use of arable lands and pesticides as in the first-generation. In contrast, the third and fourth generations of biofuels explore the utilization of microorganisms like microalgae as Schizochytrium sp., and Scenedesmus quadricauda, bacteria like Arthrobacter sp., and fungi like Mortierella isabellina [[5], [6], [7]] as alternatives to food crops. This approach is favored due to the ease of handling microorganisms and their simplified cultivation processes [8].

Tolypothix sp. is a filamentous aquatic cyanobacteria Fig. 2 collected from Bolonha lake in Pará state. It belongs to Nostocales order in which the representative cells can present special structures like akinetes and heterocysts which allow the cell to carry out exchanges with the environment such as nitrogen fixation [9]. This ability of the lineage Tolypothrix sp. turns into a promising source of nutrients and bioproducts as pigments and fatty acids [10].Fig. 2 Collection lake of Tolypothrix sp. CACIAM 22.

Fig. 2

The microalgae bioproducts market is constantly growing with an amount of USD 3.4 billion globally in 2020 with a calculated escalation of USD 4.6 billion by 2027 [11], one of the biggest obstacles faced in obtaining the biomass of these microorganisms is the cheapening in the cultivation process. When discussing biodiesel production using biomass from cyanobacteria and microalgae as raw materials, it should be noted that obtaining enough biomass remains one of the major obstacles to commercializing this biodiesel. Research efforts are focused on reducing the costs associated with biomass acquisition. Currently, there are no reports on large-scale production of biodiesel from microalgae biomass. However, in 2020, Branco-Vieira et al. [12]economically assessed the feasibility of biodiesel production using biomass from Phaeodactylum tricornutum, achieving a volume of 1811 tons of biomass and 171,705 tons of biodiesel per year. They estimated the cost at 2.01£ kg-1 for biomass production and 0.33£ L-1 for biodiesel production. This assessment revealed that in 2020, biodiesel production from microalgae biomass was still costly but manageable, with potential cost reduction strategies such as utilizing residual biomass for other high-value products like pigments and nutritious animal feed. Zhu et al. [13] demonstrated that costs could be reduced by scaling up production to 10,000 tons per year, achieving approximately US$0.60 kg-1 for raceways and US$0.47 kg-1 for photobioreactors production.Thinking about this impasse, different methodologies have already been applied to try to achieve cost reduction, among them, the use of different carbon sources, in addition to inorganic carbon from atmospheric CO2, has been common for the supplementation of the medium in which the cyanobacteria are growing [4].

Examples of materials used to enhance biomass production and add value to residual materials include the utilization of wastewater and tailings containing carbon, such as sugarcane bagasse and glycerol. These materials are used to increase biomass production due to their composition being composed of lignin, cellulose and hemicellulose. This cellulosic material produced by the decomposition of the lignocellulosic source also needs to be broken down into sugars for use by microalgae, thus maximizing resource efficiency and providing added value to the waste materials. There is a large estimate of improvement in the local market when adding value to agro-industrial waste, such as Brazil nut shells, and with a commitment to improving waste production with sustainability in mind [[14], [15], [16], [17]].

In Brazil, the extractive economy has been heavily based on the export of traditional food products such as nuts, açaí, and regional fruits. According to data released by the Brazilian Institute of Geography and Statistics (IBGE), the northern region of Brazil produced approximately 33.000 tons of Brazil nuts (Bertholletia excelsa S.B.H) in 2021, which are highly sought after due to their richness in proteins and lipids [18]. The consumption of these nuts generates a significant amount of waste in the form of their shells, which make up 90 % of the fruit and do not have any commercial use for the region [19]. In this context, due to their composition being rich in lignocellulosic material, there is great potential in utilizing them as a source of sugars for the cultivation of microorganisms.

In this way, this work sought a strategy to obtain cyanobacteria biomass using an ecofriendly carbon source derived from residual lignocellulose biomass (LCB) aimed at fatty acids production.

This research intended to investigate the Tolypothrix sp. strain CACIAM 22 potentials as a feedstock to biodiesel production. The primary objective was to evaluate the lipid production of this strain for potential use in biodiesel production. Additionally, the influence of supplementation in the culture medium using residues of Brazil nut bark from Pará as a carbon source was investigated.

2 Material and methods

A flowchart illustrating the stages of the research can be seen in Fig. 3.Fig. 3 Fluxogram of the steps to obtention of the hydrolyzed, the lipids and the fatty acids.

Fig. 3

2.1 Evaluation of cultivation conditions for biomass increase and lipid accumulation

The strain used in the study was selected from the Amazon Collection of Cyanobacteria and Microalgae (CACIAM), which was provided by the Biomolecular Technology Laboratory (LTB) research group.

The parameters of fatty acid profile, cyanobacterial growth, and biomass production were evaluated in the BG-11 culture medium, this evaluation included comparing the growth of cyanobacteria in medium without supplementation and with supplementation of glycerol and hydrolysate of Brazil nut bark (HBNS) [20,21]. Additionally, the accumulation of lipids of Tolypothrix sp. was investigated in both media with and without nitrate source after growth in the supplemented medium [22,23]. Biomass growth analyses were conducted over a thirty-day cultivation period to determine the optimal concentration of medium supplementation.

2.1.1 Biomass

A growth curve was constructed to observe the growth behavior of Tolypothrix sp. CACIAM 22 based in the dry weight. The culture was maintained under a 13-h light and 11-h dark photoperiod at 25 ± 2 °C, in triplicate. The initial biomass was standardized for all samples, using 5 ml of culture. They are harvest every 2 days for 12 days following the method of Chatsungnoen and Chisti [24] with modifications by Aboim et al. [22]. The biomass was centrifuged at 4000×g for 10 min and dried at 80 °C for 24 h, cooled in a desiccator and then weighed. All these data were used to calculate dry biomass concentrations and to construct the growth curve.

2.1.2 Supplementation

2.1.2.1 Glycerol

As one of the most common carbon substrates and models for biorefinery, glycerol has been tested as supplementation for biomass accumulation [25]. A 0.2M glycerol solution was prepared and diluted into five different concentrations (Control, 0.1M, 0.05M, 0.01M, and 0.005M) according to Cerón-Garcia [26]. These concentrations were used in 250 mL cultures to assess the growth and increase in cell biomass production over a period of thirty days. The dry weight of the biomass was determined through gravimetry. All experiments were conducted in triplicate to ensure reliable results, and the average biomass values were analyzed.

2.1.2.2 Brazil nutshell hydrolyzed

To obtain sugars for supplementation in the growing medium, Brazil nutshell were obtained from the Ver-o-Peso market. The shells were washed thoroughly with running water and then dried in an oven at 80 °C for 8 h. After drying, the shells were ground using a knife mill and subjected to particle size separation using 1 mm sieves [27].

After the physical breaking of the shells, its different components, namely cellulose, hemicellulose, and lignin, were fractionated using organic solvents as Rodríguez-Padrón work. In this step, 6g of powdered chestnut shells were mixed with a solution of ethanol and distilled water in a 1:1 ratio (25 ml:25 ml) in a bench reactor. The mixture was heated to 210 °C and agitated for 1 h and 30 min. After this process, two phases were obtained. The solid phase contained cellulose with the desired sugars, which was filtered and dried in an oven at 40 °C for 12 h and reserved for subsequent hydrolysis. The liquid phase consisted of lignin and hemicellulose and was discarded [28].

The hydrolyzed of Brazil nutshell (HBNS) was obtained by acid hydrolysis of the fractionated cellulose of the Brazil nut bark. The acid hydrolysis process followed the protocol described by Zhang et al. [29]. A 4 % sulfuric acid solution was prepared and mixed with cellulose at a ratio of 1:10 (g. mL−1). The mixture was then autoclaved at 125 °C for 30 min. After the hydrolysis step, the liquid phase was recovered for further analysis. The concentration of free reducing sugars in the liquid phase was determined using the dinitrosalicylic acid colorimetric protocol (DNS). A standard glucose curve with concentrations ranging from 0.1 to 1 g. L−1 was used to quantify the amount of reducing sugar in the hydrolysates [30]. Based on the sugar quantification results, a volume of HBNS equivalent to 1g. L−1 was used for supplementation, as suggested in the literature [31].

2.1.3 Lipids

To induce lipid accumulation within the cyanobacterial cells, the nitrogen content in the selected medium for the experiments, BG-11, was reduced according to Aboim et al., [22]. After the biomass increase in the medium supplemented with glycerol solution, the cells were transferred to a nitrogen-depleted medium with a concentration of 1g. L−1. Calculations for biomass productivity and lipid content are performed using the following formulas, as proposed by Chatsungnoen and Chisti [24].

2.1.4 Biomass productivity (BP)

Equation (1): (Bp): CF−CIt (g L−1d−1)

Were Bp (mg/L/day), with CF final biomass concentration, CI initial concentration, and t cultivation time.Equation 2(Lc):VClB(x100)(%)

Were and LC represents lipid content, V is the volume (mL) of the chloroform extract, CL indicates the concentration of lipids in the extract (mg/mL), and B refers to the amount of dry biomass used in the extraction.

2.2 Determination of the fatty acid profile

The fatty acid composition was determined using a Shimadzu CG-201 gas chromatography apparatus coupled with a flame ionization detector (FID) and split/splitless injection. A Supelcowax capillary column of dimensions 30m × 0.32 mm x 0.25 μm was employed, and a temperature ramp program was used with a rate of 10 °C/min. The temperature settings were as follows: T1 at 80 °C/min, T2 at 180 °C/min, and T3 at 250 °C/min with a hold time of 5 min. Prior to sample injection, all samples were esterified according to the AOCS (American Oil Chemistis’ Society) Ce 2–66 standard for the preparation of methyl esters according to Aboim et al. [22].

2.3 Quality parameters of biodiesel

To evaluate the quality parameters of biodiesel, the fatty acid composition produced by cyanobacteria was recorded and inputted into the Biodiesel Analyzer version 2.2 software. This software provides numerical data on biodiesel parameters, including cetane number, oxidative stability, saturation index, iodine index, viscosity, and other relevant parameters.

3 Results and discussion

3.1 Evaluation of biomass and lipid production in supplemented medium

3.1.1 Biomass

A growth curve was constructed for monitoring the behavior of the cyanobacteria as shown in Fig. 4. Biomass was accompanied over 12-days period, during which a decline in growth was observed. On day 10, when the cyanobacteria entered the early decline phase, biomass was harvested for analysis of the lipid and fatty acids parameters analysis. At this stage, the cyanobacteria produced polyunsaturated fatty acids, which is advantageous for biodiesel production.Fig. 4 The cyanobacteria growth curve. The data are average values ± standard derivation of triplicate culture.

Fig. 4

For this analysis, the experiments were planned to be conducted in two stages: one for achieving superior cell multiplication responses and the other for lipid accumulation. Initially, the cyanobacteria were cultured in a medium supplemented with HBNS and glycerol to promote cell multiplication. Subsequently, the cultivation conditions were modified to induce lipid accumulation. The collection of the final biomass and the synthesis of biodiesel took approximately 20 days to complete.

Starting from glycerol as a commonly used supplement in research, the addition of HBNS showed similar results to glycerol in terms of acting as a carbon source and influencing the increase in cyanobacterial biomass production, as observed in Table 1. Over a period of 10 days of cultivation, the wet biomass obtained from HBNS supplementation exhibited an approximately threefold increase (ranging from 3.8g to 12.7g), while supplementation with glycerol resulted in a biomass increase of around 3.5 times (ranging from 2.6g to 9.3g). The control culture displayed a twofold variation in biomass during the cultivation period (ranging from 2.5g to 5g). When compared to other sources in the table, the HBNS and glycerol showed as an excellent carbon substrate for biomass production. In this context, it should be noted that the dry weight technique should be complemented with other analyses due to the potential presence of dead cells among all cells, in order to gain a better understanding of the actual increase in biomass with the use of supplements in the culture medium.The presence of inhibitors from broken lignocellulosic biomass is still a challenge in the biomass production however, in this work, with the organic solvent pretreatment, the sugars present in the hydrolysate have increased the biomass of Tolypothrix sp.Table 1 Biomass production from Tolypothrix sp. CACIAM 22 and other sources.

Table 1Specie	Control (g.L−1)	Glycerol (g.L−1)	HBNS (g.L−1)	Other Sources	Reference	
Tolypothrix sp.	5	9.3	12.7		This Work	
Phaeodactylum tricornutum	1.4	2.9	-		[20]	
Chlorella pyrenoidosa	1.0	1.2	-		[32]	
Thalassiosira pseudonana	0.5	0.6	-		[25]	
Coelastrella sp. M-60	30.6	-	-	32.6 (LCB)	[21]	
Scenedesmus obliquus	1.1	1.5	-	-	[33]	
A. platensis	2.3	4.2	-	-	[34]	
Scenedesmus obliquus	3.4	3.5	-	4,07 (LFAH)	[35]	
C. Vulgaris	1.9	2.2	-	-	[36]	
S. incrassulatus	2.8	4.2	-	-	[37]	
Phaeodactylum tricornutum	0.8	1.2	-	-	[38]	
Scenedesmus obliquus	0.3		-	0.4 (WW)	[39]	
Scenedesmus obliquus	0.1			0.5 (Biogas)	[40]	
Chlorella sorokiniana CY-1	1.0	1.6	-	-	[41]	
Chlorella vulgaris FSP-E	1.5			1.7 (FW)	[42]	
Chlorella sp. GN1	1.7			2.1 (Light)	[43]	
Chlorella vulgaris	0.4			2.8 (VW)	[44]	
Rhodopseudomonas palustres	2.1	-	-	2.8 (Light)	[45]	
Scenedesmus dimorphus	0.6	-	-	1.4 (Glucose)	[46]	
Scenedesmus dimorphus	1.0	1.3	-	1.9 (Glucose)	[47]	
Scenedesmus dimorphus	1.0	-	-	1.2 (Sodium Acetate)	[47]	
LCB: Hydrolysate of lignocellulosic biomass. LFAH: Lipid-free algal hydrolysate. WW: Wastewater. FW: Foos Waste. Light: Flat plate reactors with different thicknesses. VW: Vinegar Wastewater.

Lin and Wu [48] investigated the impact of glycerol on the cultivation of Chlorella sp. Y8-1 for a duration of 10 days and observed a threefold increase in cell production, resulting in approximately 45 g. L−1 of dry biomass. This growth phenomenon can be attributed to the conversion of glycerol into glyceraldehyde-3-phosphate, a substrate that can be utilized in glycolysis and the pentose phosphate pathway. Similarly, Morais et al. [49] reported a fivefold enhancement in the initial biomass of Spirulina sp. LEB18 when cultured with glycerol supplementation. Jeong Choi and Whan Yu [50] examined the effects of 5g. L−1 of glycerol in the cultivation of Scenedesmus sp. and observed a 60 % higher yield compared to cultures without supplementation, reaching a value of 1.9 g. L−1.

In the cultivation supplemented with HBNS, 1g. L−1 of the hydrolysate derived from the lignocellulosic structure of Brazil nut bark was added. It was observed that, similar to glycerol, HBNS can participate in the synthesis of various metabolites. This is attributed to the presence of sugars in the HBNS structure, which can serve as substrates in different biosynthetic pathways within the cell.

3.1.2 Lipids

The lipid content (LC) of the cyanobacterium Tolypothrix sp. CACIAM 22 was evaluated in the presence of glycerol and HBNS in the culture medium. The supplementation of glycerol resulted in a 1-fold increase in lipid production (28.9 %), while the presence of HBNS led to a 2-fold increase (40.6 %), as indicated in HBNS Table 2.Table 2 Lipid content and productivity.

Table 2	Control (%)	Glycerol (%)	Suplemmentation (%)	Nitrogen decreases (mg.L−1.day−1)	Reference	
Tolypothrix sp. CACIAM 22	21.2	28.9	40.6 (HBNS)	131.2	This Work	
Limnothrix sp. CACIAM 25	18.1	–	58.3 (N-)	11.1	[22]	
Desmodesmus sp.	20	–	40 (N- Waste Water)	–	[23]	
Dictyococcus sp. VSKA18	30	–	44 (N-)	14	[21]	
Coelastrella sp. M − 60	35	–	52(N-)	5	
(N-): Culture with low levels of Nitrogen.

The increase in lipid productivity observed when HBNS was present in the medium can be attributed to its effect on the multiplication of cyanobacterial biomass rather than directly influencing the production of lipid content within the cells. It can be inferred from these results that the hydrolysate did not have a direct impact on the lipid production of the Tolypothrix sp. strain. However, when the strain was exposed to a reduced nitrogen source (1g. L−1), there was a significant increase in lipid production, with the lipid content ranging from 1.3 % to 3.4 % and a remarkable improvement of lipid productivity by approximately 9 times, with values ranging from 14g. L−1 to 131.2g. L−1.

In the study conducted by Moussa et al. [51] the cultivation of Picochlorum sp. in a nitrate-free medium resulted in a lipid productivity of 86.9 mg L−1 day−1. Similarly, Muto et al. [52] observed an increase in lipid productivity in Botryosphaerella sudetica, reaching 47.3 mg L−1 day−1 of lipid productivity and 28 % of lipid content when exposed to nitrogen deprivation during cultivation. Feng et al. [43] also investigated the cultivation of Chlorella sp. GN1 with the suppression of the nitrate source and observed an increase in lipid content from 36 % to 48 %. These studies validate the use of nitrate source suppression as a technique to induce lipid production in microalgae and cyanobacteria.

The restriction of the nutrient nitrate is widely used to regulate lipid production in cyanobacteria, as it is believed that the absence of this nutrient induces cellular stress and triggers a survival response in the cells, leading to the production of lipids as energy reserves.

3.2 Fatty acids composition

The results obtained from the 20 and 30-day cultivation with glycerol showed a biomass approximately 20 times higher than the initial biomass. However, when considering its application for biodiesel production, prolonged cultivation periods became unfavorable due to the increased production of polyunsaturated fatty acids (C18:2 and C18:3) in the cyanobacterial composition.

The fatty acid profile of the cyanobacterium Tolypothrix sp. CACIAM 22 naturally consists of approximately 36 % saturated fatty acids, with the predominant production of palmitic acid (C16:0), and 50 % unsaturated fatty acids, primarily oleic acid (C18:1). There is also a small presence of polyunsaturated linoleic acid (C18:2), which is undesirable in biodiesel composition due to its high instability. All other sources compared to this work showed similar results with little or no one polyunsaturated fatty acid, reinforcing the necessity of the absence of these fatty acids in a good quality biodiesel. Thus, the major fatty acids observed in the microalgae and cyanobacteria evaluated, were palmitc acid (C16:0), estearic acid (C18:0), and oleic acid (C18:1), showing an important characteristic of these microorganisms as sources to produce biodiesel. This information is represented in Table 3.Table 3 Fatty acids profile from different sources.

Table 3	%				
Control	Glycerol Supplementation	HBNS Supplementation	Anabaena variabilis MBDU 013	Calothrix sp. MBDU 701	Nostoc punctiforme MBDU 009	
Lauric acid (12:0)	0.6	0.6	4.9	3.4	13.3	1.4	
Miristic acid (C14:0)	0.3	3.2	1.4	-	1.7	-	
Pentadecanoic acid (C15:0)	0.1	2.4	2.2	-	-	-	
Palmitic acid (C16:0)	26.5	32.1	33.1	26.8	25.4	3.7	
Estearic acid (C18:0)	10.2	18.2	21.5	4.7	1.5	33.1	
Araquidic acid (C20:0)	1.4	4.6	5.9	-	2.6	2.1	
Behenic acid(C22:0)	0.9	0.5	-	-	6.1	23.3	
Oleic acid(C18:1)	49.6	20.5	17.5	3	-	0.9	
Linoleic acid(C18:2)	6.9	5.1	2.5	2.3	-	0.3	
Linolenic acid(C18:3)	0.4	0.5	2	1.9	-	0.5	
References	This Work	This Work	This Work	[53]	[54]	[53]	

Cultivating the cyanobacteria in the presence of glycerol or HBNS in the culture medium led to a more balanced composition of saturated and unsaturated fatty acids. The dominant fatty acids remained as palmitic acid (C16:0) and oleic acid (C18:1), which are favorable for biodiesel synthesis. This observation suggests that the Tolypothrix sp. CACIAM 22 strain has a promising potential for producing fatty acids suitable for biodiesel production. The composition results, as shown in Table 3, support this conclusion. Further analysis and optimization of cultivation conditions can enhance the biodiesel production potential of this cyanobacterial strain.

The fatty acid profile of Tolypothrix sp. CACIAM 22 in the presence of glycerol or HBNS as medium supplements allowed for the evaluation of the biodiesel quality synthesized from its biomass. Table 3 shows that the presence of saturated and unsaturated fatty acids results in a biodiesel of good quality parameters.

With the fatty acid profile, it's possible observe a biodiesel with high oxidative stability, with values ranging from 23 to 28 h. The iodine index values fell within the expected range of 27–31 g I2100 g−1fat. Additionally, the cetane number met both national and international biodiesel quality standards, with a value of 65. These findings indicate that biodiesel produced from Tolypothrix sp. CACIAM 22 has desirable characteristics and meets the required quality parameters for commercial use, see in Table 4.Table 4 Fuel properties from different sources.

Table 4Cyanobacterial Strains	Standards for biodiesel	CN	IV (g I2100 g−1 fat)	OS (hr)	References	
	Biodiesel Standard EN14214	≥51	≤120	≥6	-	
	Biodiesel Standard ASTMD6751-02	≥47	-	≥6	-	
Tolypothrix sp. Control		59.9	59.1	18.5	This Work	
Tolypothrix sp. Glycerol Supp.		65.5	31.5	23.2	This Work	
Tolypothrix sp. HBNS Supp.		65.5	27	28.3	This Work	
Anabaena variabilis MBDU 013		69	30.9	6.6	[53]	
Nostoc punctiforme MBDU 009		68.2	29.6	6.8	[53]	
Calothrix sp. MBDU 701		51	34.8	6.5	[54]	
Synechocystis sp. CACIAM05		83.1	20.5	-	[22]	
Microcystis aeruginosa CACIAM08		121	5.2	-	[22]	
Pantanalinema rosaneae CACIAM18		74.5	23.8	-	[22]	
Limnothrix sp. CACIAM25		72.1	30.1	-	[22]	
S.dimorphus		54.6	113.3	-	[46]	
DunaliellasalinaKSA-HS022		53.9	112.6	-	[55]	
Micractinium reisseri		60.4	39.5	-	[56]	
Parachlorella kessleri		53.5	122.2	-	[57]	
Dunaliella salina		48.1	111.7	-	[58]	
S. incrassatulus		62	-	19	[59]	
Scenedesmus obliquus		-	70	-	[60]	
Chlorella pyrenoidosa		61.6	76.1	-	[61]	
Chlorella pyrenoidosa (FACHB- 9)		51.6	138.3	3.7	[55]	
Chlorella protothecoides		51	-	6	[62]	
Nodosilineanodulosa SNMVBTAR001		-	115.2	-	[63]	
Chlorella sp. AARL G049		46.8	113.8	6.6	[64]	
Tetradesmus obliquus AARL G090		56.5	72	11.8	[64]	
Desmodesmus opoliensis AARL G085		56.3	71.7	11	[64]	
CN: Cetane Number. IV: Iodine Value. OS: Oxidative Stability.

The presence of high oxidative stability in biodiesel indicates low chemical reactivity and enhanced stability. This characteristic makes the biodiesel more resistant to the effects of the external environment, such as temperature, heat, and light, when stored and exposed [65]. Consequently, biodiesel with high oxidative stability retains its quality for a longer duration, exhibiting minimal degradation or deterioration under adverse conditions. For Tolypothrix sp. CACIAM 22 was observed to have over 18 h of oxidative stability according to Biodiesel Analyzer simulation. This attribute serves as an indicator of a high-quality biodiesel product when compares to standard of UE and ASTM, and which maintains its desired properties and performance over time, ensuring reliability and suitability for various applications [53,65].

The confirmed parameters validate that the optimal balance between saturated and unsaturated fatty acids in Tolypothrix sp. biomass composition yields superior results in assessing biodiesel quality. This fatty acid composition corroborates the results observed in other studies as the Sohedein et al. [66], in which the presence of saturated fatty acids improves the stability of the biodiesel produced and, however, the presence of monounsaturated fatty acids balances the quality of this biodiesel, not allowing the condensation and crystallization of these acids. at room temperature. This harmony is advantageous to produce high-quality biodiesel with excellent characteristics.

The obtained values of saturated and unsaturated fatty acids in this study are consistent with those reported by Kumar and Sharma [67], who observed 46 % saturated fatty acids and 53 % unsaturated fatty acids in a Tolypothrix strain they investigated. The balance of saturated fatty acids observed in this study increased when the biomass was supplemented, suggesting its influence on the generation of saturated fatty acids and a decrease in the production of polyunsaturated fatty acids. This finding is in line with the work of Bansal et al. [68], who reported that glycerol influences the increase in saturated fatty acid production when used in the cultivation of Rhodotorula mucilagenosa IIPL32. Similarly, Ashokkumar et al. [69] obtained similar results concerning the biodiesel quality produced by the cyanobacterium Synechocystis sp., with a cetane number of 52.5, iodine index of 88, and low acidity with a value of 0.5 (mm2. s−1). These findings indicate a biodiesel with low unsaturation levels and quality equivalent to commercially available biodiesel.

Due to the great influence that the use of fossil sources generates in the increase of environmental problems in the world, the accumulation of lipids in the biomass of microalgae and cyanobacteria such as Tolypothrix sp. proves to be an excellent alternative for a world that prioritizes the use of less aggressive natural resources and that contributes to the reduction of climate change in the world [70] in fact, the market tends to reach the global mark of US$ 15 billion by 2030 according to www.strategyr.com (accessed May 17, 2024), with a compound annual growth rate (CAGR) increasing 7.7 % in 2023–2030. However, obtaining an excellent amount of cyanobacterial biomass is still a challenge to increasing industrial use, thus, combining with residual carbon sources would be a path to overcome these questions.

Hence, the microalgal biomass diminishes the toxic gas release in the atmosphere while using residual lignocellulosic has advantages in being transformed into a carbon source and decreasing the accumulation of wastes. Nowadays, the reuse of lignocellulosic waste like HBNS has a big value to the environment because of the distinct products generated from it, and for the maintenance of the bioeconomy that takes advantage, valuing the residual matter with different bioproducts [71,72].

4 Conclusion

In this study, the cyanobacterium Tolypothrix sp. CACIAM 22 was investigated for a duration of 20 days to assess the impact of supplementation with Brazil nut bark hydrolysate (HBNS) on biomass, lipid, and fatty acid production for biodiesel synthesis. During the period in a medium supplemented with HBNS, the biomass accumulation increased by approximately 3.5 times. Subsequently, in a medium with reduced nitrate source (1g. L−1), there was a 3 % increase in lipid content and a remarkable 9-fold increase in lipid productivity in biomass, reaching a value of 131.2g. L−1. Supplementation with HBNS resulted in a different balance in the composition of saturated and unsaturated fatty acids, with higher quantities of palmitic acid (C16:0) and oleic acid (C18:1) then, biodiesel derived from the biomass of cyanobacteria showed excellent quality parameters, including high oxidative stability (around 28 h), a cetane number close to 65, and an iodine index of approximately 27 (g I2 100 g−1 fat).

Thus, this study has confirmed the potential for recovering valuable bioproducts from the biomass of the cyanobacterium Tolypothrix sp. CACIAM 22, including its use in the production of third-generation biodiesel as a substitute for conventional vegetable-based raw materials. It is still necessary to study ways to improve the costs of obtaining lipids from cyanobacteria biomass and turning it into a competitive feedstock to produce biodiesel.

Therefore, this investigation can still contribute to the development of a biorefinery approach using residual Brazil nutshell biomass as a carbon source for maximizing the utilization of the biomass and its resources.

CRediT authorship contribution statement

Deborah Terra de Oliveira: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Rutiléia de Jesus Paiva: Writing – review & editing, Methodology. Vanessa Albuquerque de Mescouto: Writing – review & editing, Methodology. Sara Roberta Ferreira da Silva: Writing – review & editing, Conceptualization. Ana Alice Farias Da Costa: Writing – review & editing, Visualization. Agenor Valadares Santos: Writing – review & editing, Methodology, Data curation. Evonnildo Costa Gonçalves: Writing – review & editing, Methodology, Data curation. Geraldo Narciso da Rocha Filho: Resources. Renata Coelho Rodrigues Noronha: Resources. Luís Adriano Santos do Nascimento: Writing – review & editing, Resources, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgments

The authors would like to thank CNPQ under grant number 315279/2021-4 , BASA 2022/233 , 10.13039/501100005288 FAPESPA /CNPQ/10.13039/501100002322 CAPES (015/2023 and 073/2023 ), 10.13039/501100002322 CAPES (PhD. Fellowship for Vanessa Mescouto, Rutileia Paiva, Ana Costa and Deborah Terra), 10.13039/100017425 PROPESP/UFPA and the laboratories that supported this work: The Laboratory of Research and Analysis of Fuels (LAPAC/10.13039/501100007382 UFPA ) and the Laboratory of Oils of the Amazon Oil Laboratory, Brazil, Laboratory of Enzyme Biotechnology and Biotransformations (LABEB) and Laboratory of Biomolecular Technology (LTB).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36343.
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