Fumaric acid production from fermented oil palm empty fruit bunches using fungal isolate K20: a comparison between free and immobilized cells

Fungi isolation

A total of 26 soil samples were collected from Bangkok and Nakhon Ratchasima provinces in Thailand separately in plastic bags, taken to a laboratory at Kasetsart University in Bangkok, stored at 4 °C, and analyzed within 24 h of collection. Field experiments were conducted under the principles and ethical standards approved by the National Research Council of Thailand (NRCT) and Thailand Science Research and Innovation (TSRI), (Approval reference: MHESI 6390.FB. 6.1/1/2564).

One gram soil sample was suspended in sterile 0.85% saline solution to prepare serial dilutions spread in triplicate on potato dextrose agar (PDA) plates. The plates were then incubated at 30 °C for 48–168 h. The fungal isolates were selected based on morphological characteristics, sub-cultured using PDA slants, and stored at 4 °C.

Screening for organic acid-producing microorganisms

Fungal isolates were inoculated on a mineral agar acid indicator medium containing 120 g/L glucose, 3.02 g/L (NH₄)₂SO₄, 0.25 g/L MgSO₄·7H₂O, 0.04 g/L ZnSO₄·7H₂O, 0.15 g/L KH₂PO₄, 20 g/L agar, 0.2 g/L bromocresol green, and 1.5 mL/L Triton X-100 in distilled water (pH: 5.5; Suntornsuk & Hang, 1994). Fungal isolates that form a yellow zone around the colony, indicating their ability to produce acid, were selected and tested for FA production.

Screening for FA production

The fungal isolates were cultured on PDA plates at 30 °C for 7 days. The spores were then washed twice with sterile water to obtain a spore suspension. After filtration through sterile cotton layers, the spore suspension was counted under a microscope using a hemocytometer (Boeco Germany, Hamburg, Germany) and diluted to the desired concentration. For the seed culture, 1 mL spore suspended in distilled water (1 × 10⁷ spores/mL) was inoculated into the growth medium containing 30 g/L glucose, 0.6 g/L KH₂PO₄, 0.0176 g/L ZnSO₄·7H₂O, 1.5 g/L urea, 0.5 g/L MgSO₄·7H₂O, 0.0005 g/L FeSO₄·7H₂O (pH: 7). After incubating for 24 h, the cell pellet was washed with autoclaved distilled water and fermented in the production medium containing 90 g/L glucose, 0.6 g/L KH₂PO₄, 0.04 g/L ZnSO₄·7H₂O, 0.05 g/L urea, 0.5 g/L MgSO₄·7H₂O, 1.25 g/L (NH₄)₂SO₄, 0.1 g/L Na₂CO₃ (pH: 5), in a static condition at 30 °C for 168 h. The samples were aliquoted every 24 h for determining the FA concentration. The experiment was done in triplicate.

Fungal isolate identification

Genomic DNA was extracted from the mycelia using an E.Z.N.A. Forensic DNA kit (Omega Bio-tek Inc., Norcross, GA, USA), following the manufacturer’s instructions. The DNA purified using this kit was suitable for polymerase chain reaction (PCR) analysis.

The internal transcribed spacer (ITS) region was amplified in a 50-mL reaction mixture (Applied Biosystems, Foster City, CA, USA) containing 1 U Taq DNA polymerase, 1 × buffer, 2.5 mM MgCl₂, 0.2 mM dNTPs, and 0.2 µM each primer (ITS5 and ITS4). All the primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The PCR conditions were pre-denaturation at 96 °C for 2 min; 35 cycles of denaturation at 96 °C for 1 min, annealing at 53 °C for 1 min, and extension at 72 °C for 1.5 min; final extension at 72 °C for 10 min. The purified amplified genomic DNA was electrophoresed in ethidium bromide-stained 1% agarose (Fluka Biochemika, Gillingham, UK) and observed under ultraviolet light. The amplified and purified genes were sequenced in both directions using an automated DNA sequencer (Macrogen Inc., Seoul, Korea). Nucleotide sequences obtained from all primers were assembled using the Cap Contig Assembly Program v.3 (Huang & Madan, 1999), BioEdit Program v.7.2.5 (a biological sequence alignment editor program; Hall, 1999). The sequences were compared with nucleotide sequences in GenBank, CBS, or a suitable database.

Raw material and reference microorganisms

This study used oil palm EFB sourced from the Suksomboon Palm Oil Industry (Chonburi Province, Thailand) as the raw material. The EFB were sun-dried, ground to obtain approximately 2.5 cm particles, and then stored in a sealed plastic bag until further use.

Two commercial strains, R. oryzae TISTR 3535 and R. arrhizus TISTR 3198 (NRRL 1469), were procured from the Thailand Institute of Scientific and Technological Research (TISTR) and used as reference strains. These strains were cultured and maintained on PDA agar at 4 °C until they were ready for use.

Raw material pre-treatment

Approximately 200 g oil palm EFB was subjected to steam explosion at 20 MPa (210 °C) for 4 min in a 2.5-L stainless steel batch digester (Nitto Koatsu Co. Ltd., Tokyo, Japan) (adapted from Siramon, Punsuvon & Vaithanomsat, 2018). The material was then separated into a solid residue and liquid by filtration through a cheesecloth. The solid residue was soaked in hot water at 80 °C for 60 min, washed with tap water until it reached a neutral pH, and further delignified.

The steam explosion-pretreated materials were delignified by soaking in 15% (w/v) NaOH and incubated at 90 °C for 30 min. Subsequently, the suspension was filtered and the solid residue was washed with tap water until it reached a neutral pH. The delignified solid was air-dried and stored in a sealed plastic bag for further separate hydrolysis and fermentation (SHF).

Separate hydrolysis and fermentation (SHF)

The steam-exploded and delignified oil palm EFB were individually used for FA production via SHF. The raw material was enzymatically saccharified using a Cellic CTec 2 (185FPU/mL; Novozyme A/S, Basgsværd, Denmark) in a citrate buffer (50 mM, pH 4.8) at 50 °C and 150 rpm for 24 h. The enzyme loading was fixed at 15 FPU/g of raw material. The glucose concentration was quantitatively determined using a high-performance liquid chromatography column (HPLC, Shimadzu LC-20A; Shimadzu, Kyoto, Japan). Briefly, 100 mL enzymatic hydrolysate with initial oil palm EFB-derived glucose of 3.5% was used for FA fermentation in a 250-mL Erlenmeyer flask supplemented with the seed culture and production medium, except that the enzymatic hydrolysate was used instead of glucose. Static fermentation was performed at 30 °C for 168 h. The samples were aliquoted every 24 h for determining the FA concentration.

Comparison of immobilized and free cells

To compare the efficiency of free and immobilized cells for FA production, the fungal isolate was prepared in either free or immobilized form. For the free cells, 1% spores (1 × 10⁷ spores/mL) were inoculated to a 250-mL flask containing 100 mL growth medium, and then incubated at 30 °C and 150 rpm for 24 h. The cells were then harvested, washed twice with sterile distilled water, and transferred into production medium. For the immobilized cells, a 0.5 mm × 0.5 mm × 0.5 mm polyurethane sponge was used as a support for the cell immobilization process. The polyurethane cube was placed in a 250-mL Erlenmeyer flask containing 100 mL growth medium and inoculated with the fungal spore suspension. After incubation for 24 h in a rotary shaker at 30 °C with 150 rpm, the growth medium was removed. The immobilized cells were washed twice with sterile water, transferred to the production medium, and cultured at 30 °C and 150 rpm in the rotary shaker for 96 h. The samples were aliquoted every 24 h to determine the FA concentration.

Batch fermentation in an air-lift fermenter

Batch fermentation was conducted in a 3-L air-lift fermenter (MCI-6C; B.E. Marubishi Co. Ltd., Bangkok, Thailand) with 2 L working volume with 80, 100, and 120 g/L oil palm EFB-derived glucose instead of pure refined glucose as the substrate. The fermentation condition was fixed at 30 °C, initial pH of 3.5, and 1.5 volume of gas per volume of liquid per minute (VVM) aeration rate (Boondaeng et al., 2022). The samples were aliquoted every 24 for 120 h to determine the FA concentration using high-performance liquid chromatography (HPLC).

Analytical methods

An oil palm EFB sub-sample was milled using a Wiley mill (Kinematica AG Co., Ltd., Tokyo, Japan) to 40-mesh particles. Its chemical composition was quantitatively determined according to the TAPPI standard methods: TAPPI T204 om-88 (1997) for extractives; TAPPI T211 om-93 (2002) for ash; TAPPI T222 om-88 (1988) for Klason (acid-insoluble) lignin; TAPPI T223 cm-84 (2001) for pentosan; and TAPPI T203 om-88 (1992) for α-cellulose. An HPLC with an Aminex HPX-87H column (300 × 7.8 mm²; Bio-Rad, Richmond, USA) was used to measure FA concentration quantitatively. The mobile phase was 0.005 NH₂SO₄ at 40 °C and 0.6 mL/min flow rate.

The glucose concentration was quantitatively determined a high-performance liquid chromatography column (HPLC, Shimadzu LC-20A; Shimadzu, Kyoto, Japan) consisting of a refractive index detector (RID). The analyses were separated on an Aminex HPX-87P column at 80 °C. The HPLC system operated at a flow rate 0.6 mL/min in deionized water as the mobile phase (Liu et al., 2010).

Fungal sample isolation and identification

In total, 463 fungal isolates were obtained from 26 soil samples, of which 15 isolates significantly produced FA (Table 1). Isolate K20 produced the maximum FA (3.25 g/L) after 96 h of cultivation, while the reference strains R. oryzae TISTR 3535 and R. arrhizus TISTR 3198 produced 0.12 and 0.09 g/L FA, respectively. After confirming its ability to produce FA, isolate K20 was selected for further investigation. The morphological characteristics of the isolate K20 are shown in Fig. 1A. Initially, an isolate K20 colony growing on PDA appeared white and cottony. Over time, it became heavily speckled with sporangia, gradually turning blackish-gray, and spreading rapidly to the substrate with stolons through the rhizoids (Fig. 1A). The globose to ovoid single-celled sporangiophores, originating from the rhizoids within a spherical structure known as the sporangium, are straight, smooth-walled, and responsible for producing numerous multinucleated spores (Fig. 1B). Based on its morphological and molecular characteristics, isolate K20 closely resembled R. arrhizus. The identity of isolate K20 was confirmed by amplifying and sequencing the ITS region using ITS1. The isolate was placed within a clade comprising R. arrhizus reference isolates (90.31%). The phylogenetic tree constructed using the NJ method showed that isolate K20 was closely related to the subclade R. arrhizus (Fig. 2). Isolate K20 was 90.31% similar to R. arrhizus. Based on the morphological characteristics and the phylogenetic tree, isolate K20 exhibits characteristics that suggest that it may warrant classification as a new genus, a matter that requires further investigation. The ITS gene sequence of this isolate has been deposited in the GenBank database under the accession number OR717492.

Chemical composition of oil palm EFB

The chemical compositions of the oil palm EFB before and after steam explosion pre-treatment are listed in Table 2. The cellulose content of oil palm EFB before and after steam explosion pre-treatment was 65.48% and 75.25%, respectively. In contrast, the percentages of hemicellulose, lignin, and extractive in ethanol/benzene decreased from 22.19%, 15.18%, and 3.21% to 2.02%, 7.50%, and 2.11%, respectively, after the steam explosion pre-treatment. The cellulose content of other materials, such as Acacia mangium, Acacia hybrid (Boondaeng et al., 2015), sugarcane bagasse (Guilherme et al., 2015), and corncobs (Liu et al., 2010) is 44%, 42%, 38.6%, and 38.8%, respectively, before pre-treatment. The cellulose content of pre-treated A. mangium, A. hybrid, sugarcane bagasse, and corncobs was 76%, 74%, 65%, and 65.7%, respectively.

Comparison of immobilized and free cells

Immobilization techniques have been widely explored to increase the possibility of a continuous fermentation process, as well as reuse microbial cells for several cycles on an industrial scale (Naude & Nicol, 2017; Deng & Aita, 2018; Swart, Brink & Nicol, 2022). The amount of FA produced after fermentation for 96 h with free and immobilized isolate K20 cells was 3.25 and 1.50 g/L, respectively. Thus, free cell fermentation had a higher volumetric productivity (0.034 g/L/h) than immobilized cell fermentation (0.016 g/L/h).

Batch fermentation in an air-lift fermenter

A 3-L air-lift fermentation was used to upscale FA production. Oil palm EFB-derived glucose was used instead of commercial glucose at the initial concentrations of 80, 100, and 120 g/L. The highest FA concentration (44 g/L) was obtained within 72 h of fermentation with 100 g/L initial oil palm EFB-derived glucose (Fig. 3). The yield and productivity were 0.44 g/g and 0.61 g/L/h, respectively, which were 13.54-fold higher than those in a volumetric flask. These results indicate that isolate K20 can produce FA from lignocellulosic material-derived glucose hydrolysate.