Fermentation of acidic-pretreated glycerol for enhanced 1,3-PDO production by immobilized Clostridium butyricum JKT 37 on coconut shell activated carbon

doi:10.21203/rs.3.rs-4072494/v1

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Research Article

Fermentation of acidic-pretreated glycerol for enhanced 1,3-PDO production by immobilized Clostridium butyricum JKT 37 on coconut shell activated carbon

https://doi.org/10.21203/rs.3.rs-4072494/v1

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published 14 Oct, 2024

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Bioproduction of 1,3-propanediol (1,3-PDO) is regarded as a low carbon footprint bioprocess with a 33% reduction of greenhouse gas (GHG) emissions compared to conventional chemical processes. In line with Sustainable Development Goal (SDG) 12, bioproduction of 1,3-PDO closes the loop between biodiesel waste glycerol and biobased 1,3-PDO to establish a circular bioeconomy. There are limited studies on the bioconversion of biodiesel-derived glycerol into 1,3-PDO via the immobilized cell biocatalysis route. In this study, the production of 1,3-PDO was enhanced by the wild-type Clostridium butyricum JKT 37 immobilized on the activated carbon of coconut shell (CSAC) as supporting material using the acidic-pretreated glycerol as a carbon source. Among various mesh sizes of CSAC tested, the 6-12 mesh immobilizer had enhanced cell density by about 94.43% compared to the suspended cell system. Following the acidic pretreatment in 37% (v/v) HCl, the pretreated glycerol had 85.60% glycerol with complete removal of soaps. The immobilized cell fermentation using pretreated glycerol produced 8.04 ± 0.34 g/L 1,3-PDO with 0.62 ± 0.02 mol/mol of yield, 15.81% and 27.78% higher than the control, respectively. Five repeated batches of immobilized cell fermentation had resulted in the average 1,3-PDO titer, yield, and productivity of 16.40 ± 0.58 g/L, 0.60 ± 0.03 mol/mol, and 0.68 ± 0.02 g/L.h, respectively. The metabolism pathway gradually shifted to a reductive branch when immobilized cells were reused in repeated batch fermentation, proven by the reduction in organic acid formation and the increased ratio of 1,3-PDO-to-total organic acids.

Pretreated glycerol

3-propanediol

immobilized Clostridium butyricum

coconut shell activated carbon

immobilizer reusability

With the increasing awareness of the disastrous consequences brought by excessive exploitation and usage of non-sustainable fossil fuels, the world is urged to develop a new supply chain of sustainable fuels and chemicals. Bioenergy which corresponds to Sustainable Development Goal 7: Affordable and Clean Energy is a mutual long-term climate goal around the world. Biodiesel, regarded as a renewable and biodegradable fuel, has become one of the alternative energy in the transportation field (Decarpigny et al. 2022). By 2050, biodiesel production will inevitably rise by 0.5%, or equivalent to 130,000 barrels per day (Chilakamarry et al. 2021). The production of this low carbon-emission fuel has gradually transformed the world's energy system, mitigating consequences brought by fossil fuels and expected to meeting the high demand for cleaner and sustainable energy.

Despite its benefits in increasing energy security and improving air quality, the production of biodiesel via the transesterification of animal fats or vegetable oils with short-chain alcohols, would leave behind waste glycerol as the primary byproduct, accounting for around 10% of the total fuel produced by weight (Sittijunda et al. 2020). In Southeast Asia, especially Indonesia, Malaysia and Thailand, the mandatory palm oil-based biodiesel blending rates are as high as 35%, 10% and 7%, respectively (Nupueng et al. 2018; Wirawan et al. 2024; Zulqarnain et al. 2020). In line with the United Nations’ Sustainable Development Goal (SDG) 12: Responsible Production and Consumption, implementing a circular waste management system in palm oil-based biodiesel industries via conversion of waste glycerol into valuable 1,3-propanediol (1,3-PDO) might establish a circular bioeconomy. Recently in year 2023, the price of biodiesel-derived glycerol has dropped by about 50% as supply surges in the region. There are several applications of crude glycerol such as combustion, poultry-feeding, composting, and thermochemical/biological conversions to value-added commodity (Brown 2007; Johnson et al. 2007; Lammers et al. 2008; Lee et al. 2015).

The environmental issue has accelerated the development of waste glycerol management, where one of the effective methods is to valorize this underutilized material via biological means (Chilakamarry et al. 2021). Biodiesel-derived waste glycerol that contains around 60 to 80% glycerol could be converted to biochemical via microbial fermentation, avoiding the use of solvent chemicals in the conventional chemical route. Waste glycerol could be converted into several high-valued biochemical compounds such as 1,3-propanediol (1,3-PDO) (Hazimah et al. 2003; Samul et al. 2014). 1,3-PDO is in increasing demand in a myriad of applications. By comparing the environmental performance, the biobased PDO has a lower greenhouse gas (GHG) emissions of 2.5 to 6.7 kg CO₂eq/kg PDO compared to the fossil-based PDO (4.04 to 9.4 kg CO₂eq/kg PDO) that primarily produced from the hydroformylation of ethylene oxide (Stegmann 2014; van Heerden et al. 2023). In fact, 1,3-PDO could be produced from glycerol fermentation by wild-type bacteria, for instance, the genera of Clostridium (Lan et al. 2021; Wang et al. 2020), Klebsiella (Kim et al. 2021; Laura et al. 2020), Citrobacter (GÜNGÖRMÜŞLER 2021; Maina et al. 2019). Among the 1,3-PDO-producing strains, Clostridium spp. is the most exploited strict anaerobe that can produce 1,3-PDO as a major product from glycerol consumption with an appreciable yield of 0.59 to 0.71 mol/mol, along with the coproduction of other biochemical compounds such as butyric acids and acetic acids (Tey et al. 2023).

Crude glycerol, e.g. as generated from the biodiesel plant, contains a variety of contaminants such as residual methanol, fatty acid methyl/ethyl ester, soaps, free fatty acids (FFAs), and matter organic non-glycerol (MONG) (Laura et al. 2020; Wu et al. 2016). The contaminants could be an inhibiting agent that affects the morphology and metabolism of the bacteria. Hence, pretreatment of the crude glycerol can minimize the inhibition effects of substrate during the fermentation (Laura et al. 2020). Many studies reported the enhanced fermentation performance using pretreated glycerol as a carbon source compared to the crude glycerol (Loureiro-Pinto et al. 2016; Wilkens et al. 2012). Several studies discovered that using crude glycerol gave higher 1,3-PDO titer, yield, and productivity without inhibiting microbial fermentation (Jun et al. 2010; Rodriguez et al. 2016). The tolerance level of the impurities in crude glycerol differs among the 1,3-PDO-producing strains and should be investigated to combat the glycerol glut problem, and to ensure the maximum bioconversion of glycerol to valuable 1,3-PDO. Besides, the initial glycerol concentration is a crucial factor in determining the highest 1,3-PDO yield. A high or low substrate concentration in the fermentation medium could affect the cell metabolism and substrate diffusion (Yang et al. 2018).

One of the major bottlenecks of suspended cell fermentation is the low cell culture density or metabolite productivity. Since 1,3-PDO is a product associated with cell growth, intensifying cell density has become the primary determinant of effective 1,3-PDO synthesis. The synthesis of 1,3-PDO is an important cellular response for initiating vital metabolic pathways for microbial development in addition to balancing the intracellular redox potential (Gonen et al. 2012). The use of immobilized cells on solid supports provides numerous bioprocess advantages, such as elevating cell density, protecting bacteria from harsh conditions, accelerating substrate uptake rate and product formation rate (Gungormusler-Yilmaz et al. 2016). There are a few solid supports that have been evaluated for bacterial immobilization to produce 1,3-PDO production, i.e. pumice stone (Gonen et al. 2012), ceramic rings (Gungormusler et al. 2011), ceramic balls (Gungormusler et al. 2011), polyurethane foam (PUF) (Casali et al. 2012), glass beads (Gonen et al. 2013), glass raschig rings (Gonen et al. 2013), polyvinyl alcohol (PVA) hydrogel (Dolejš et al. 2019), keramsite (Kowalska et al. 2017; Mizielińska et al. 2020), modified polyurethane foams (PUFs) (Mizielińska et al. 2020), and alginate beads (Loureiro-Pinto et al. 2017; Yang et al. 2017).

Among the immobilization techniques, adsorption has gained substantial attention as it is the easiest yet effective approach to maintaining enzyme activity (Mehrotra et al. 2021; Zdarta et al. 2019). The cell adsorption on solid supports is governed by Van der Waals forces and ionic interactions. Activated carbon is excellent support for immobilized cell systems as this inert material can sustain the bacteria viability and the colonization density (Jamali et al. 2017). Coconut shell activated carbon (CSAC) has been used as the support material in producing succinic acid by Actinobacillus succinogenes 130Z (Luthfi et al. 2017) and biohydrogen via Thermoanaerobacterium thermosaccharolyticum sp. (Jamali et al. 2017). CSAC derived from the agroindustrial residue, is highly available and affordable with its exceptional adsorption properties such as well-developed pore structure and high surface area compared to other activated carbon available in the market (Lee et al. 2021; Luthfi et al. 2017).

The adhesion capacity and cohesion ability of the supporting materials highly affect the performance of cell immobilization. Surface area determines the cell-surface interaction whilst porous properties imply the cell-cell interaction. These two indicators, as characterized by surface morphology (Brunauer–Emmett–Teller (BET) surface area and pore characteristics), are vital in selecting a suitable immobilizer for microbial fermentation (Luthfi et al. 2017). The surface morphology and microstructure of the CSAC solid carrier before and after immobilization can be visualized using Field emission scanning electron microscopy (FESEM). The ability to reuse immobilized cells with stable bioprocess performance could significantly reduce fermentation costs, shorten total fermentation time, and simplify the downstream processing (Yang et al. 2017; Zhang et al. 2018). A repeated batch strategy, in which fermented broth is removed and replaced with a new fermentation medium can be implemented to evaluate the reusability of the immobilized cells.

This paper aims to evaluate the performance of the bioconversion of acidic-pretreated (crude) glycerol to 1,3-PDO as catalyzed by the wild-type C. butyricum JKT 37 immobilized on CSAC as the solid support to increase the yield and productivity of 1,3-PDO. Analyses of the CSAC surface before and after cell immobilization were characterized, by FESEM for the visualization of surface morphology and BET analysis for pore diameter, volume and surface area were conducted. A range of fermentation, using the immobilized bacteria to catalyze pure (control), crude (control), and acidic-pretreated glycerol in producing 1,3-PDO in the suspended or immobilized bacteria system, was conducted where their respective titer and yield were compared and discussed. The optimum initial substrate concentration which gave the highest yield of 1,3-PDO was selected for the reusability study of the immobilized cells in five cycles of fermentation.

2.1 Acid pretreatment of crude glycerol

Crude glycerol used in this research was provided by a Malaysian company that manufactures palm-based biodiesel. Crude glycerol is pretreated to remove those impurities using the pretreatment process by Tan et al. (2018) which involves heating, acidification, microfiltration, and finally neutralization. The crude glycerol in solid form at room temperature was pre-heated at 60°C in a water bath until liquefied and turned into a dark brown color as a step to remove methanol due to its low volatility. Hydrochloric acid (HCl) (37% (v/v)) was added to the liquefied crude glycerol until a pH of 4.5 to 5.0. The acidic pretreatment was conducted under a fume hood with ventilation where the excess soaps were eliminated by transforming into free fatty acids (FFAs). The crude glycerol turned into a yellowish liquid with blackish oil droplets floating on the surface. The blackish oils developed clumps and hardened on the surface of glycerol due to cooling. A vacuum pump was used to filter the mixture from the fatty matter using 0.22 µm nylon filter paper. The filtrate was neutralized to pH 7 using 2 M potassium hydroxide (KOH) and stored as pretreated glycerol under 4°C until used (Tan et al. 2018).

2.2 Characterization of glycerol samples

The physicochemical properties of the pure, crude, pretreated glycerol were evaluated including the pH, density, glycerol content, soap content, free fatty acids (FFAs), moisture content, and ash content.

The pH of glycerol was measured at room temperature using a Professional Benchtop pH Meter (BP3001) by dissolving 1 g of glycerol sample in 50 mL of deionized water. The density was determined using a pycnometer based on the method by Nanda et al. (2014). The volume of the pycnometer was determined by dividing the difference between the mass of the filled and dried pycnometer by the density of water. The dry pycnometer was filled with a glycerol sample at the same temperature and the mass was recorded. The density of the glycerol samples was determined by dividing the mass of the sample by the volume of the pycnometer.

The glycerol content in glycerol samples was analyzed by the UltiMate 3000 high-performance liquid chromatography (HPLC, Thermo Scientific, USA). One gram of the sample was dissolved in 50 mL of water, and 2 mL of the solution was filtered through a 0.22 µm syringe filter and injected into the HPLC vial. The sample was analyzed using the Phenomenex RoA 300 mm x 7.8 mm column set at 60 ºC and refractive index detector (RID) set at 40 ºC, through elution of the mobile phase (5 mN H₂SO₄ solution) isocratically at a flow rate of 0.6 mL/min. An established plot of the chromatogram area versus standardized glycerol concentration was used to determine the mass percentage of glycerol in the samples.

FFA was determined using the titrimetric method according to the standard method of AOCS Official Method Ca 5a-40 (AOCS Reapproved 2009). The soap content in glycerol samples was analyzed using the titrimetric method described in ISO 1615–1976(E) (ISO 1976). The moisture content of in the glycerol samples was determined following the method mentioned by Laura et al. (2020) where a 10 g sample was heated at 105°C until a constant weight. Ash content was analyzed using the standard method ISO 6884:2008 by burning 10 g of sample in a muffle furnace at 550°C for 3 h (ISO 2010).

2.3 Preparation of microbial inoculum and fermentation media

The wild-type C. butyricum JKT 37 used for the fermentation of glycerol was isolated from a wastewater treatment plant of a palm oil mill effluent (POME) located in Malaysia as developed by Tee et al. (2017). The stock cultures are maintained in 40% glycerol at -80°C. To prepare the microbial inoculum, the bacterium was revived by inoculating 1 mL of glycerol stocks into 49 mL of Reinforced Clostridial Medium (RCM) medium contained in 160 mL of anaerobic serum bottle at 37°C and 120 rpm over 12 h. The fermentation media used in this study comprises of (per L of water): K₂HPO₄, 3.4 g; KH₂PO₄, 1.3 g; (NH₄)₂SO₄, 2.0 g; MgSO₄·7H₂O, 0.2 g; CaCl₂·2H₂O, 0.02 g; yeast extract, 1.0 g; L-cysteine HCl, 0.5 g; Fe solution, 1 mL; and trace elements solution, 2 mL. The Fe solution contains FeSO₄.5H₂O, 5 g/L and 37% (v/v) HCl, 4 mL/L. The trace elements solution consists of (per L of water): ZnCl₂, 70 mg; MnCl₂·4H₂O, 100 mg; H₃BO₃, 60 mg; CoCl₂·6H₂O, 200 mg; CuCl₂·2H₂O, 20 mg; NiCl₂·6H₂O, 20 mg; Na₂MoO₄·2H₂O, 40 mg.

The pure, crude, or pretreated glycerol samples was added into the fermentation medium as the sole carbon source in a separate batch of microbial fermentation. Crude glycerol was heated to obtain a liquefied sample, and its pH was adjusted to around pH 8 using 37% (v/v) HCl prior to fermentation. Magnesium carbonate (MgCO₃) was used to control the pH of the medium throughout the fermentation.

2.4 Cell density improvement in fermentation using different mesh sizes of CSAC as immobilizers

The wild-type C. butyricum JKT 37 as isolated in section 2.1 was immobilized on different mesh sizes of CSAC as purchased from Concepts Ecotech (Selangor, Malaysia). The effect of different CSAC mesh sizes as the best solid support for bacterial immobilization was investigated. RCM medium was used in determining the overall cell density improvement of the immobilized bacteria of 4 mesh sizes (6–12, 10–20, 20–40, 60–100). Another RCM medium without an immobilizer was prepared as the control for the calculation of cell density improvement. The solid supports were washed with deionized water and dried in an oven overnight at 50°C. 5% (w/v) loading of immobilizer of different mesh sizes was added to 49 mL of RCM in the respective 160 mL serum bottles, purged with 99.99% nitrogen, and sterilized. The glycerol stocks of C. butyricum JKT 37 were inoculated into the serum bottles. The incubation was carried out at 37°C, 120 rpm for 48 h to ensure the maximum consumption of nutrients by the bacteria. After immobilizing the cells, the broth and immobilized bacteria were filtered through vacuum filtration using 0.22 µm filter paper. The supports with adsorbed cells was washed with distilled water. The residues were dried in an oven at 50°C until a constant weight. The dry cell weight (DCW) of the suspended (control) or immobilized fermentation expressed in g/L was determined through the gravimetric method and subsequently analyzed for cell density improvement, where the difference in DCW of the immobilized and suspended system was divided by the DCW of the suspended cell (Zhou et al. 2018).

2.5 Batch fermentation of suspended and immobilized cell

CSAC of 6–12 mesh was chosen as the immobilizer for all types of glycerol fermentation to compare with the suspended cell fermentation based on a cell density improvement study as described in Section 2.4. The solid supports were loaded at a 5% (w/v) ratio in the medium following nitrogen gas purge and sterilization, where the cell immobilization process and fermentation and were executed simultaneously. 10% (v/v) of the cell culture (section 2.3) grown to the exponential phase was transferred into 160 mL serum bottles containing 45 mL of fermentation medium by a sterile syringe. The batch fermentation was conducted at 37 ºC, 120 rpm agitation speed for 24 h. The cell culture samples were periodically collected to determine the concentration of glycerol, 1,3-PDO, acetic acid and butyric acid. The results reported as mean ± standard deviations are obtained from two independent experiments.

2.6 Reusability study of the immobilized cell fermentation at an optimum initial substrate concentration

The initial concentration of pure glycerol ranging from 20 to 60 g/L was tested in the suspended fermentation to determine the optimum yield of 1,3-PDO. The fermentation was conducted based on the same method described in section 2.5 using the medium composition as described in section 2.3. The samples were collected at the beginning and the end of the fermentation process analyzed by HPLC.

To examine the reusability of the immobilizer, the optimum initial pure glycerol concentration which gave the highest yield of 1,3-PDO was selected for five repeated batches of fermentation using pretreated glycerol as the substrate to produce 1,3-PDO. The repeated batch of fermentation was initiated as a batch with a 100-mL working volume of fermentation broth in a 300 mL serum bottle and 24 h of cultivation. The actively growing inoculum size of the initial batch culture was 10% (v/v). The fermentation pH was maintained using 10 g/L MgCO₃. The culture temperature and agitation speed were maintained at 37 ºC and 120 rpm, respectively. After 24h, the fermented broth was withdrawn from the serum bottle and supplemented with an equal volume of fresh sterile anaerobic fermentation medium (same composition as that utilized to begin batch culture as described in section 2.3). The empty-and-fill protocol was repeated until the five cycles of fermentation (Kaur et al. 2012). The results reported as mean ± standard deviations are obtained from two independent experiments.

2.7 Analytical methods

A gas adsorption analyzer (Micromeritics 3Flex 3500, Norcross, GA, USA) was applied in determining the Brunauer–Emmett–Teller (BET) surface area and pore characteristics of carrier materials using the N₂ adsorption/desorption isotherm at -196°C (Luthfi et al. 2017).

Surface visualization of the immobilized C. butyricum on CSAC was carried out using Field Emission Scanning Electron Microscopy (FESEM) (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Raw CSAC was freeze-dried in a freeze dryer (Labconco, Kansas City, MO) at -50°C for 24 h. The immobilized carriers were fixed with 2.0 wt% glutaraldehyde at 4°C overnight and were then washed with 0.1 M phosphate-buffered saline (PBS) at pH 7.2 for 30 min. Subsequently, the samples were subjected to dehydration through successive immersion in 30–100% (v/v) ethanol to prevent samples from damage. The dehydrated samples were further dried using a critical point dryer (Leica EM CPD 300, Wetzlar, Germany). The dried solid supports with immobilized cells and freeze-dried raw carrier were then subjected to sputter coating with platinum and further analysis using FESEM (Luthfi et al. 2019).

The concentration of substrate (glycerol), product (1,3-PDO) and byproduct metabolites including for acetic acid and butyric acid in the fermentation broths were analyzed using HPLC as described in section 2.2. The substrate and metabolite concentration were determined using the standard calibration curve of the respective pure component. The conversion yield of 1,3-PDO \(\left({Y}_{P/S}\right)\) in mol product (P)/mol substrate (S) was determined as the ratio of the P concentration to the relative S (glycerol) consumption. The productivity of 1,3-PDO \(\left({R}_{p}\right)\) and the rate of glycerol consumption \(\left({R}_{S}\right)\), expressed in g/L.h, were determined by dividing the 1,3-PDO and glycerol concentration by their relative fermentation time. The glycerol utilization efficiency (%) is computed by dividing the glycerol consumed with the initial glycerol concentration.

3.1 Characterization of glycerol samples

Due to high pH and room-temperature insolubility, crude glycerol is unsuitable for direct use in fermentation media (Tan et al. 2018). Several pretreatments were therefore introduced to get rid of its contaminants. Table 1 presents the physicochemical properties of pure and crude glycerol, and the pretreated glycerol.

Table 1

Physicochemical properties of different types of glycerol.
Properties	Pure glycerol	Crude glycerol	Pretreated glycerol
pH	7.09 ± 0.02	11.64 ± 0.02	6.96 ± 0.01
Density (g/cm³)	1.26 ± 0.00	1.22 ± 0.00	1.24 ± 0.00
Moisture (% w/w)	1.56 ± 0.40	6.49 ± 0.19	11.18 ± 0.01
Ash (% w/w)	N.D.	3.70 ± 0.02	3.81 ± 0.02
Glycerol content (% w/w)	99.50 ± 0.00	75.52 ± 0.38	85.60 ± 0.44
Soap (% w/w)	N.D.	21.23 ± 0.13	N.D.
FFAs (% w/w)	N.D.	N.D.	0.05 ± 0.00
Total composition	101.06	106.94	100.64

Crude glycerol has a high pH of 11.64 and around 75.52% of glycerol content; its alkaline nature and impurities content (e.g. soaps, and FFAs) make it inappropriate to be directly used as a carbon source in microbial fermentation. The high pH is due to the use of alkali catalysts in the transesterification process of biodiesel production. Hence, pretreatment was implemented to increase the glycerol content and remove the contaminants. Through acidification, 21.23% of soap in crude glycerol was converted into FFAs, which were later removed through microfiltration and then neutralization using KOH. The pretreated glycerol contains 85.60% glycerol and 0.05% FFAs, indicating a significant removal of soap. The pretreated glycerol has a final pH of 6.96, which is close to the optimum pH for fermentation, where the final density was higher than that of crude glycerol owing to the removal of soaps. The increase in moisture content (4.69%) of the pretreated glycerol is due to water production during the neutralization process. Ash in the crude and pretreated glycerol, which are 3.70% and 3.81%, respectively, is produced mainly by the KOH catalyst used during the transesterification or biodiesel process (Nanda et al. 2014).

3.2 Cell density improvement by immobilized C. butyricum JKT 37

Improvements in cell density by immobilized C. butyricum JKT 37 were evaluated using four mesh sizes of the solid supports of CSAC with the respective characteristics shown in Table 2.

Table 2

Characteristics of CSAC at different mesh sizes.
	CSAC mesh sizes
Characteristics	6–12	10–20	20–40	60–100
BET surface area (m²/g)	748.42	791.00	1059.57	864.95
Micropore area (m²/g)	665.51	675.48	878.51	685.43
Total pore volume (cm³/g)	0.32	0.35	0.45	0.40
Micropore volume (cm³/g)	0.26	0.27	0.35	0.27
Proportion of micropore volume (%)	81.25	77.14	77.78	67.50
Pore diameter (nm)	1.68	1.76	1.70	1.85

The BET-surface area increases when particle size decreases except for the CSAC of 60–100 mesh. This phenomenon is similar to the study of Hodson (1998), Zareba et al. (2016), and Zhang et al. (2017) where the BET-surface area is nearly independent of the particle size but mesopores and/or macropores become more dominant as size decreases. As shown in Table 2, all mesh sizes of CSAC have a higher volume of wide micropores and the existence of small mesopores, as indicated by a high proportion of micropore volume (approximately 68–83% of total pore volume) (Hidayu et al. 2013). The average pore type of CSAC is microporous and mesoporous. The pore diameter for the studied CSAC mesh sizes in this research ranges from 1.68 nm to 1.85 nm, as analyzed using the BET method. In general, smaller carrier pores might experience clogging issues when there is concentrated flow or cell buildup despite having a larger surface area. The transfer of nutrients from the pores to the cells would be restricted (Woo et al. 2022). A greater pore size or a higher proportion of mesopores might hinder the attachment of microbes as the high shear force does not encourage easy colonization (Jamali et al. 2017). Even though the highly porous structure and high surface area of CSAC represent the ability of cell retention, the differences in surface area, pore volume, and pore diameter at each carrier cause certain limitations in the immobilization such as the limited mass transfer of nutrients into pores, cell leaching, and clogging.

Since the formation of 1,3-PDO is growth-associated, the increase in cell concentration indicates improved cell growth, enabling a higher rate of 1,3-PDO produced during fermentation. A successful immobilization can be represented by the enhanced DCW and cell density improvement. Figure 1 illustrates the cell density improvement using solid supports at varying mesh sizes. The DCW of the suspended C. butyricum JKT 37 without solid supports was 2.29 ± 0.21 g/L.

Using cell density achieved in the suspended fermentation as the basis or control, the improvement in density of the immobilized cells at different CSAC mesh size was determined. According to the bar chart in Fig. 1, the cell density improvement decreased from 94.43–52.54% when the mesh sizes of CSAC increased from 6–12 mesh (largest) to 60–100 mesh (smallest). This can be attributed to the smallest pore size of the 6–12 mesh solid support. As mentioned earlier in this section, despite having higher surface area, the three larger mesh and pore sizes of CSAC (10–20, 20–40, and 60–100), encountered a lower cell density improvement owing to the higher energy collision between the cells to the carrier surface. A similar phenomenon was observed in a study by Jamali et al. (2017) where they reported a lower degree of cell growth using larger pore sizes (1.06–1.09 nm) of the solid support. The smallest pore diameter (1.68 mm) and highest micropore volume proportion (81.25%) of 6–12 mesh CSAC used in this study is optimal to immobilize bacteria effectively, hence this mesh size was selected in the subsequent study of fermentation performance to maximize the cell density.

3.4 Surface morphology visualization of immobilized C. butyricum JKT 37 on CSAC

FESEM was applied to provide visual insights into the surface morphology and microstructure of CSAC as a cell carrier before and after immobilization. Figure 2 (a) represents the FESEM image of raw CSAC that is irregular and highly porous. This postulates that numerous holes and cavities can serve as the space for bacterial growth and protection from the environment, particularly, the fermentation broth.

There are three possible mechanisms of the cell adsorption, which are cell aggregation, adsorption on the solid surface via hydrogen, covalent, ionic bonding and cell-matrix hydrophobic interaction, and lastly adsorption in the carriers’ cavities and colony development in macropores (Agudelo Escobar et al. 2012; Anita et al. 2016). FESEM images have confirmed the immobilization of C. butyricum JKT 37 on/into the CSAC through the adsorption mechanism. Figure 2 (b) and (c) are close-up views of CSAC, which depict the presence of pores and crevices, the intrinsic features of CSAC that serve as the spaces for cell growth. Figures 2 (d) and (e) captured by FESEM revealed the impregnated CSAC after immobilization, where the rod-shaped C. butyricum JKT 37 attached to the rough surfaces and grown within the crevices of porous CSAC. Each cell has an average diameter and length of 0.5 µm and 2–3 µm respectively (Zhou et al. 2018). Hence, the bacteria can move and grow in the crevices and even infiltrate the small pores of CSAC. The cells tend to form chain-like structures or interspersed within the CSAC.

Based on Fig. 2 (f), the formation of an extracellular component (thin white thread) between C. butyricum JKT 37 bacteria and CSAC surface was observed through FESEM, implying the successful immobilization of Clostridium spp. on the support carriers. The porous structure of CSAC helps to maintain cell vitality and allows microbial colonization.

3.5 Fermentation of pure glycerol fermentation by suspended and immobilized C. butyricum JKT 37

To assess the fermentation performance of the immobilized cell, batch fermentation of the pure glycerol in both the suspended and immobilized cell on CSAC to produce 1,3-PDO production, were conducted and compared. The kinetic parameters of the fermentation are tabulated in Table 3.

Table 3

Kinetics parameters of the batch fermentation of suspended and immobilized cells using pure glycerol at 9 h.
Fermentation	Suspended cells	Immobilized cells
Glycerol consumed (g/L)	9.22 ± 0.10	15.09 ± 0.09
1,3-PDO (g/L)	3.73 ± 0.62	6.40 ± 0.88
Acetic acid (g/L)	0.97 ± 0.16	1.03 ± 0.35
Butyric acid (g/L)	1.09 ± 0.05	1.02 ± 0.04
AA/BA ratio	0.89	1.02
Glycerol utilization efficiency (%)	51.95 ± 0.02	97.20 ± 0.01
\({\varvec{R}}_{\varvec{S}}\) (g/L.h)	1.02 ± 0.01	1.68 ± 0.01
\({\varvec{Y}}_{\varvec{P}/\varvec{S}}\) (mol/mol)	0.46 ± 0.12	0.51 ± 0.07
\({\varvec{R}}_{\varvec{p}}\) (g/L.h)	0.41 ± 0.07	0.71 ± 0.18

The time course profile of both suspended and immobilized systems under the same conditions is presented in Fig. 3 which exhibits the residual glycerol and metabolite concentrations.

The utilization efficiency of glycerol by the immobilized cells reached 97.20% at 9 h, elucidating a faster fermentation speed with an accelerated substrate consumption rate due to the higher cell growth. At 9 h of fermentation as assisted by the immobilized cells, there was almost no residual glycerol found in the broth with a high consumption rate of glycerol (1.68 ± 0.01 g/L.h). In contrast, the suspended cell fermentation was left with a high concentration of residue glycerol (9.22 ± 0.10 g/L) that was consumed at a low rate of 1.02 ± 0.01 g/L.h when the fermentation was carried out after 9 h. A higher concentration of of 1,3-PDO (6.40 ± 0.88 g/L) was achieved by immobilized cell fermentation with a molar yield of 0.51 ± 0.07 mol/mol, which was higher than the suspended cell system (3.73 ± 0.62 g/L, 0.46 ± 0.12 mol/mol) as shown in Table 3. Moreover, the 1,3-PDO productivity of the CSAC immobilized cell system was 0.71 ± 0.18 g/L.h, i.e. 1.71 times higher than that in the suspended cell culture. It can be concluded that cell immobilization had augmented the productivity and accelerated product production rate, which has greatly reduced the fermentation time. With the immobilization verification of FESEM images, a favorable extracellular microenvironment in CSAC has promoted cell proliferation and adsorption, subsequently ameliorating the fermentation performance.

3.6 Fermentation of different glycerol by suspended and immobilized C. butyricum JKT 37

The pure, biodiesel-derived crude and pretreated glycerol was used as the carbon source in the respective batch fermentation to compare the performance of the immobilized C. butyricum JKT 37. CSAC was incorporated into the fermentation as the immobilizer to enhance cell density. Table 4 summarizes the fermentation results of both the suspended and immobilized cell fermentation using three types of glycerol after 24 h.

Table 4

Experimental results of suspended and immobilized fermentation using different types of glycerol at 24 h.
Fermentation	Suspended			Immobilized
Glycerol	Pure	Crude	Pretreated	Pure	Crude	Pretreated
Initial glycerol (g/L)	18.46 ± 0.44	20.00 ± 0.20	19.55 ± 0.65	15.21 ± 0.09	15.86 ± 0.34	16.07 ± 0.13
1,3-PDO (g/L)	5.42 ± 1.17	5.31 ± 1.15	6.94 ± 0.41	6.59 ± 0.15	6.28 ± 0.28	8.04 ± 0.34
Acetic acid (AA) (g/L)	0.54 ± 0.13	0.58 ± 0.05	0.67 ± 0.03	1.07 ± 0.02	1.10 ± 0.02	1.35 ± 0.20
Butyric acid (BA) (g/L)	2.92 ± 0.64	2.60 ± 0.72	3.32 ± 0.58	1.37 ± 0.17	1.16 ± 0.16	1.47 ± 0.12
Total acids (AA + BA) (g/L)	3.46 ± 0.78	3.17 ± 0.77	4.00 ± 0.55	2.44 ± 0.19	2.27 ± 0.19	2.81 ± 0.09
AA/BA ratio	0.19	0.22	0.20	0.78	0.95	0.92
Glycerol utilization efficiency (%)	79.93 ± 1.99	90.68 ± 0.20	86.62 ± 2.89	99.33 ± 0.67	99.24 ± 0.76	99.06 ± 0.97
\({Y}_{P/S}\) (mol/mol)	0.45 ± 0.09	0.36 ± 0.04	0.48 ± 0.02	0.53 ± 0.01	0.48 ± 0.03	0.62 ± 0.02
Final pH	5.24 ± 0.04	5.21 ± 0.01	5.23 ± 0.01	6.08 ± 0.03	6.03 ± 0.02	6.02 ± 0.09

Suspended cell fermentation of the pretreated glycerol achieved a higher titer (6.94 ± 0.41 g/L) and yield (0.48 ± 0.02 mol/mol) of 1,3-PDO as compared to the other two fermentation using pure and crude glycerol as the substrate. After applying immobilized cells on CSAC, the fermentation of the pretreated glycerol achieved an even higher titer (8.04 ± 0.34 g/L) and yield (0.62 ± 0.02 mol/mol) than the other two fermentation batches with pure and crude glycerol.

Suspended cell fermentation of the crude glycerol achieved the lowest titer (5.31 ± 1.15 g/L) of 1,3-PDO possibly due to the contaminants in the crude glycerol, e.g. soap, salts and/or MONG. The respective low yield (0.36 ± 0.04 mol/mol) obtained reveals the significant inhibitory potential of the crude glycerol in microbial fermentation using crude glycerol as the substrate. The double bond in soap may be the inhibitor influencing the growth behavior of the bacteria (Wang et al. 2019). The high amount of soap in the crude glycerol (Table 1) induced a negative impact on bacterial growth and assimilation, which resulted in a low 1,3-PDO yield, similar to the study of Laura et al. (2020) which used crude glycerol containing around 22.29% (w/w) of soap. Gram-positive bacteria such as Clostridium spp. are generally more susceptible to crude glycerol (Chatzifragkou et al. 2010; Moon et al. 2010). The presence of impurities would be likely to exert inhibitory activity over 1,3-PDO synthesis by Clostridium species. This phenomenon aligns with the previously reported work (Loureiro-Pinto et al. 2016).

Nearly 100% of glycerol consumption was observed when adopting CSAC as a bacterial immobilizer in the fermentation of pure, pretreated, crude glycerol at 12 h of the fermentation. However, the suspended cell fermentation had a lower glycerol utilization efficiency, ranging from 80 to 91%, than the immobilized cell system (99+%) after 24 h fermentation. It implies that fermentation occurs more quickly in the biofilm of the immobilization cultures than in the suspended cell cultures. The high availability of biomass has likely accelerated the rate of substrate consumption and the cell protection provided by CSAC from harsh environments, such as acids produced during fermentation.

The bioproduction of 1,3-PDO from glycerol accompanies the formation of side products where the major metabolites produced after fermentation is butyric acid, followed by acetic acid. A lower acetate/butyrate ratio was discovered for the suspended cell system, which indicated a lower ATP yield (Tan et al. 2018). Thus, less energy is present for microbial growth, and consequently, the glycerol fermentation is affected. The total acids produced by the immobilized cell system are lesser than those produced by suspended cell cultivation; hence, the final pH of fermentation broth for the former is higher that of the latter as shown in Table 4. A higher acetate/butyrate ratio was found in immobilized systems is an indicator of faster microbial growth. It can be deduced that the rapid biomass growth demanded more ATP and thus, formation of acetic acid was triggered to produce more energy instead of the butyric acid pathway (Gungormusler-Yilmaz et al. 2014). The accumulation of weak organic acids resulted in an increased concentration of hydrogen ions around and inside the cell, which demands a large amount of energy to eradicate the ions from the cell by proton pumps (Szymanowska-Powalowska 2014). This implies the production of excessive organic acids will affect cell growth and inhibit the product yield, as observed in this study.

By comparing the suspended cell fermentation of different substrates, the 1,3-PDO titer and yield of pretreated glycerol are the highest, followed by pure glycerol and crude glycerol as shown in Fig. 4. The trend is similar when comparing the 1,3-PDO titer and yield of CSAC-immobilized cell fermentation using different types of glycerol. Notably, enhanced performance in 1,3-PDO production with pretreated glycerol was observed in both suspended and immobilized cells, as shown in Fig. 4. This elucidates that the simple pretreatment applied to crude glycerol removed most of the impurities, and subsequently, the pretreated glycerol can serve as a suitable feedstock in another biorefinery process. Fermentation using pretreated glycerol gave a better molar yield than pure glycerol for both suspended and immobilized cell fermentation, with 6.67% and 16.98% of improvement, respectively, where a similar phenomenon was observed in the study by Tan et al. (2018). This could be ascribed to the presence of phosphates originally in raw glycerol and salts (NaCl and KCl) after pretreatment (Yang et al. 2017). These components might act as buffer pH medium in the early stages of the fermentation, ultimately enhancing growth, substrate uptake, and production rates of 1.3-PDO (Rodriguez et al. 2016).

The adoption of CSAC as the supporting material in the fermentation of crude glycerol has elevated its titer and yield by 1.18- and 1.32-fold, respectively, as compared to its suspended cell cultivation. Both the titer and yield (6.28 ± 0.28 g/L, 0.48 ± 0.03 mol/mol) obtained are higher than that of the suspended cell fermentation with pure glycerol which is 5.42 ± 1.17 g/L and 0.45 ± 0.09 mol/mol, respectively. This revealed that the immobilizer might act as a shield for the cells, making it more tolerable to environment perturbation, which leads to glycerol consumption with minimum inhibition caused by the impurities. The composition of contaminants in the crude glycerol differs among different biodiesel manufacturers which may be affected by different oil types, catalysts, alcohols, and specialized biodiesel technologies. Therefore, the quality of the crude glycerol has a significant impact on the robustness of the fermentation performance. With the implementation of CSAC as support material, the 1,3-PDO titer and molar yield of all types of substrates were augmented from their respective suspended cell system.

3.7 Determination of optimum 1,3-PDO yield from different initial substrate concentration

The typical initial substrate concentration ranged from 20 to 60 g/L; 60 g/L of glycerol concentration is recommended in several studies to maintain the bacterial activity and prevent inhibition of 1,3-PDO production (Suratago et al. 2012; Yen et al. 2014). The initial substrate concentration ranging from 20 to 60 g/L was tested to prevent substrate inhibition and at the same time to determine the optimum 1,3-PDO yield. As the initial substrate concentration increased, the titer of 1,3-PDO also elevated, according to Table 5 and Fig. 5. This phenomenon illustrates that no inhibitory effect was observed in the fermentation by C. butyricum JKT 37 using glycerol concentration between 20 g/L to 60 g/L. A similar trend of the effect of substrate concentration towards the fermentation performance was observed in Yang et al. (2017), where the critical substrate concentration of 40 g/L was the same in this study. Nonetheless, the 1,3-PDO yield increased from 0.44 ± 0.00 mol/mol to 0.63 ± 0.01 mol/mol when the initial glycerol concentration increased from 20 g/L to 40 g/L in this study, and eventually dropped to 0.57 mol/mol when higher substrate concentrations were applied as illustrated in Fig. 5. Hence, the highest yield (0.63 mol/mol) was observed with an initial substrate concentration of 40 g/L, whereas the highest titer (23.28 ± 1.52 g/L) was obtained by an initial glycerol concentration of 60 g/L. However, the residual glycerol concentration after fermentation increased along with the increase of initial substrate concentration in the medium. This elucidates that supplying more glycerol did not further enhance the 1,3-PDO yield even though its titer was improved. The total acids produced (AA + BA) were observed in an increasing trend (Table 5) with the increase in glycerol concentration, except for the glycerol concentration of 60 g/L. In the view of 1,3-PDO/(AA + BA) ratio, 30 g/L of initial glycerol concentration has the value of 2.99, which indicates a higher production of 1,3-PDO as compared to the total organic acids, followed by the substrate concentration of 40 g/L with the value of 2.86. Regardless of the relatively higher major product-to-byproducts ratio of 60 g/L glycerol, it had the lowest substrate consumption and 1,3-PDO yield. Moreover, based on Table 5, 30g/L glycerol had the highest ratio of 1,3-PDO to butyric acid, with a value of 5.04 among the other glycerol concentrations. Hence, the substrate concentration used in repeated batch cultivation was maintained in the range of 30 g/L to 40 g/L to ensure the maximum 1,3-PDO yield to reduce the cost of raw material.

Table 5

Experimental results of batch fermentation by *C. butyricum* JKT 37 at various initial glycerol concentrations at 24 h.
Initial glycerol concentration (g/L)	20	30	40	50	60
Residual glycerol (g/L)	0.84 ± 0.03	1.39 ± 0.05	2.81 ± 0.30	4.40 ± 0.29	8.85 ± 0.66
Glycerol utilization efficiency (%)	95.93	95.39	92.86	91.20	84.89
1,3-PDO (g/L)	6.94 ± 0.29	14.58 ± 0.05	18.95 ± 0.11	21.57 ± 0.43	23.28 ± 1.52
\({\varvec{Y}}_{\varvec{P}/\varvec{S}}\) (mol/mol)	0.44 ± 0.00	0.61 ± 0.00	0.63 ± 0.01	0.57 ± 0.01	0.57 ± 0.02
Acetic acid (g/L)	1.53 ± 0.17	1.98 ± 0.16	2.16 ± 0.07	2.33 ± 0.00	1.92 ± 0.01
Butyric acid (g/L)	1.61 ± 0.54	2.89 ± 0.27	4.47 ± 0.20	6.37 ± 0.15	5.75 ± 0.17
AA + BA (g/L)	3.14	4.87	6.62	8.70	7.67
1,3-PDO/(AA + BA) ratio	2.21	2.99	2.86	2.48	3.04
1,3-PDO/BA ratio	4.31	5.04	4.24	3.39	4.05

3.8 Reusability study of the immobilized C. butyricum JKT 37

The reusability and stability of immobilized C. butyricum JKT 37 on CSAC were studied through five repeated batch fermentation carried out within 120 h, and the experimental results are summarized in Table 6.

Table 6

Reusability study of the immobilized *C. butyricum* JKT 37 by five batches of repeated fermentation.
Number of repeated batch	1	2	3	4	5
Initial glycerol concentration (g/L)	34.31 ± 0.27	35.14 ± 0.52	33.08 ± 0.38	31.68 ± 0.75	32.17 ± 0.60
Residual glycerol concentration (g/L)	0.33 ± 0.02	0.24 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Glycerol utilization efficiency (%)	99.03	99.31	100.00	100.00	100.00
1,3-PDO (g/L)	17.45 ± 0.42	15.83 ± 0.36	16.38 ± 0.62	15.91 ± 1.37	16.42 ± 0.10
\({\varvec{Y}}_{\varvec{P}/\varvec{S}}\)(mol/mol)	0.62 ± 0.01	0.55 ± 0.00	0.60 ± 0.02	0.61 ± 0.04	0.62 ± 0.01
\({\varvec{R}}_{\varvec{p}}\) (g/L.h)	0.73 ± 0.02	0.66 ± 0.02	0.68 ± 0.03	0.66 ± 0.06	0.68 ± 0.00
Acetic acid (g/L)	1.45 ± 0.18	0.75 ± 0.01	0.77 ± 0.01	0.70 ± 0.04	0.69 ± 0.07
Butyric acid (g/L)	1.60 ± 0.06	2.25 ± 0.34	2.07 ± 0.17	1.90 ± 0.06	1.75 ± 0.07
Total acids (g/L)	3.05 ± 0.12	3.00 ± 0.33	2.84 ± 0.18	2.61 ± 0.02	2.40 ± 0.09
1,3-PDO/AA + BA ratio	5.73	5.27	5.77	6.10	6.72

The initial glycerol concentration was maintained between 30 g/L to 40 g/L to ensure the maximum 1,3-PDO yield, as demonstrated by the previous section of the study. The glycerol utilization efficiency increases from the first cycle until a complete consumption of glycerol is observed, starting from the third to fifth cultivations. Figure 6 outlines the final titer, yield, and productivity of 1,3-PDO during the repeated batch fermentation with CSAC immobilized C. butyricum JKT 37 using pretreated glycerol. Figure 6 shows that the 1,3-PDO yields and productivities were stable, and no decreasing trends were observed in the repeated batch immobilized fermentation. However, the reduced 1,3-PDO yield in the subsequent cycles was observed in the study of Yang et al. (2017) who used alginate beads for cell immobilization. The average 1,3-PDO titer, yield, and productivity of the five repeated batch immobilized cultivations are 16.40 ± 0.58 g/L, 0.60 ± 0.03 mol/mol, and 0.68 ± 0.02 g/L.h.

Based on Table 6, the final concentration of acetic acid and butyric acid in the first cycle of batch culture was 1.45 ± 0.18 g/L and 1.60 ± 0.06 g/L respectively, producing similar yields. However, the accumulation of butyric acid was approximately two times higher than acetic acid, starting from the second cycle to the fifth cycle of repeated batch fermentation. It is likely that the increase in butyrate was another way to recycle or consume NADH while the energy requirements were stable or lower. In other words, NADH released from both oxidative pathways and biomass synthesis were used in the formation of 1,3-PDO and other pathways during each fermentation cycle to maintain the overall redox balance (Gungormusler-Yilmaz et al. 2014; Zhang et al. 2018). This scenario was verified by the gradual increase of the ratio of 1,3-PDO to the total organic acids produced (1,3-PDO/AA + BA) from the first to the fifth cycle of repeated batch immobilized fermentation, suggesting the desirable shift to reductive pathway metabolic pathway in producing 1,3-PDO (Khanna et al. 2013). Furthermore, a decreasing trend was observed for the accumulation of total organic acids from the first to fifth run of repeated batch immobilized fermentation. As such, it could be deduced that there is no reduction in immobilized cell mass throughout the five repeated batch cultivation, evidenced by the reduction in byproduct formation and the increased ratio of 1,3-PDO/(AA + BA) (Yang et al. 2017).

The nature of the carrier serves as the protective agent of the cell against harsh conditions such as pH, temperature, solvents, or even heavy metal in the medium (Gungormusler-Yilmaz et al. 2016). Hence, the metabolic pathway of the bacteria during fermentation will be shifted towards the reductive branch, ensuring the stable production of 1,3-PDO regardless of the high concentration of substrate and metabolites. The development of biofilm by C. butyricum JKT 37, as observed in the FESEM image protects the cells from environmental stresses, resulting in enhanced cell tolerance and 1,3-PDO production (Gungormusler-Yilmaz et al. 2016). Immobilized C. butyricum JKT 37 on CSAC showed a significant improvement even on serum bottle batch fermentation as it can achieve a competitive 1,3-PDO yield of 0.62 mol/mol, normally reported within 0.39–0.65 mol/mol in the literature (Drożdżyńska et al. 2014; Lan et al. 2021; Laura et al. 2020; Zabed et al. 2019). The immobilization of C butyricum JKT 37 is a promising strategy that allows intensification of downstream processing due to the easy removal of the immobilized biomass from the fermentation medium. Further scale-up study of the bioreactor with the better-controlled system can elevate the fermentation performance of 1,3-PDO synthesis through cell immobilization.

The titer and yield of 1,3-propanediol (1,3-PDO), as catalyzed by the fermentation of immobilized C. butyricum JKT 37 on the selected CSAC size from the four size meshes of coconut shell activated carbon (CSAC) as the solid support for three types of glycerol (pure, crude, acid-pretreated) were analyzed and compared with those obtained in the suspended cell fermentation as a control. Fermentation of the acid-pretreated glycerol provided the highest titer and yield of 1,3-PDO using the smallest mesh size of CSAC as the solid support of the cell immobilization, as compared to the suspended cell fermentation as a control, and as compared to the immobilized cells using crude glycerol as the substrate. The results concluded the significant impact of contaminant removal via acid-pretreatment of the crude glycerol to ensure enhanced production of 1,3-PDO by suppressing the total formation of the undesired organic acid byproducts (acetic acid and butyric acid). Of all four mesh sizes of the CSAC compared, the use of the smallest mesh size (6–12) as the solid support for the immobilized cells has provided the highest improvement in cell density. The enhanced surface characteristics of the immobilized cells using the best or smallest mesh size (6–12) were well supported by all the surface analyses which reveal 20.37% higher proportion of micropore volume, 9.19% smaller pore diameter, and higher colonization level as evidenced by the FESEM analyses, as compared to the lowest cell density achieved using the largest mesh size of CSAC (60–100). The reusability test of immobilized cells on CSAC revealed that at the fifth cycle, 94% of 1,3-PDO titer and productivity were maintained compared to the first cycle with the same yield of 0.62 mol/mol after five cycles of repetitive fermentation using pretreated glycerol as the substrate. This concludes that CSAC provides a stable environment for cell growth and reuse for the fermentation of waste glycerol. Overall, the yield of 1,3-PDO achieved in the immobilized cell fermentation, regardless of the type of glycerol used, were consistently higher (> 17%) as compared to those conducted using the free cells fermentation. These results concluded the significant contribution of cell immobilization on CSAC to enhance the bioconversion of glycerol to 1,3-PDO.

1,3-PDO – 1,3-propanediol, AA – acetic acid, BA – butyric acid, BET – Brunauer–Emmett–Teller, CSAC – coconut shell activated carbon, DCW – dry cell weight, EPSs – extracellular polymeric substance, FESEM – Field Emission Scanning Electron Microscopy, FFAs – free fatty acids, H₂SO₄– sulfuric acid, HCl – hydrochloric acid, HPLC – High-Performance Liquid Chromatography, KCl – potassium chloride, KOH –potassium hydroxide, MgCO₃ – magnesium carbonate, MONG – matter organic non-glycerol, N₂ – nitrogen, NaCl – sodium chloride, NaOH – sodium chloride, PBS – phosphate-buffered saline, POME – palm oil mill effluent, RCM – Reinforced Clostridial Medium, RID – refractive index detector

ACKNOWLEDGEMENTS

This material is based upon work supported by the Ministry of Science, Technology and Innovation Malaysia (MOSTI) under Technology Development Fund 1 (TeD 1) Project “TDF07211418” and Malaysia Toray Science Foundation (MTSF) Science & Technology Research Grant “220527STRG0160”. This research is also supported by Xiamen University Malaysia Research Fund (XMUMRF) Project “XMUMRF/2022-C10/IENG/0047” and “XMUMRF/2023-C12/IENG/0060”.

Ethical Approval

Not applicable.

Competing interests

The guest editor, Dr. Woon Kok Sin is involved in this research and is one of the coauthors.

Authors’ contributions

Conceptualization: KYT, JPT; Data curation: KYT; Methodology: KYT; Formal analysis and investigation: KYT, AAIL, SFAM; Writing - original draft preparation: KYT; Writing - review and editing: KYT, KSQ, CTL, SSM, JPT; Funding acquisition: KYT, JPT; Resources: AAIL, SFAM, SKY, YWH, HS, JPT; Supervision: AAIL, JPT; Project administration: JPT

Funding

This work was financially supported by the Ministry of Science, Technology and Innovation Malaysia (MOSTI) under Technology Development Fund 1 (TeD 1) Project “TDF07211418” and Malaysia Toray Science Foundation (MTSF) Science & Technology Research Grant “220527STRG0160”. This research is also supported by Xiamen University Malaysia Research Fund (XMUMRF) Project “XMUMRF/2022-C10/IENG/0047” and “XMUMRF/2023-C12/IENG/0060”.

Availability of data and materials

Data and materials of this research work will be available based on the request.

Agudelo Escobar LM, Salazar Álvarez U, Peñuela M (2012) Yeast immobilization in lignocellulosic wastes for ethanol production in packed bed bioreactor. Revista Facultad de Ingeniería Universidad de Antioquia 66-76.
Anita SH, Mangunwardoyo W, Yopi Y (2016) Sugarcane Bagasse as a Carrier for the Immobilization of Saccharomyces cerevisiaein Bioethanol Production. Makara Journal of Technology 20:4. https://doi.org/http://dx.doi.org/10.7454/mst.v20i2.3059
AOCS (Reapproved 2009) Free Fatty Acids. AOCS Official Method Ca 5a-40, American Oil Chemists' Society, Urbana, Illinois
Brown R (2007) Biodiesel co-product markets in wyoming for wyoming department of agriculture. Lakewood, co: International center for appropriate & sustainable technology.
Casali S, Gungormusler M, Bertin L, Fava F, Azbar N (2012) Development of a biofilm technology for the production of 1, 3-propanediol (1, 3-PDO) from crude glycerol. Biochemical engineering journal 64:84-90. https://doi.org/https://doi.org/10.1016/j.bej.2011.11.012
Chatzifragkou A, Dietz D, Komaitis M, Zeng AP, Papanikolaou S (2010) Effect of biodiesel‐derived waste glycerol impurities on biomass and 1, 3‐propanediol production of Clostridium butyricum VPI 1718. Biotechnology and bioengineering 107:76-84. https://doi.org/https://doi.org/10.1002/bit.22767
Chilakamarry CR, Sakinah A, Zularisam A, Pandey A (2021) Glycerol waste to value added products and its potential applications. Systems Microbiology and Biomanufacturing 1:378-396. https://doi.org/http://dx.doi.org/10.1007/s43393-021-00036-w
Decarpigny C, Aljawish A, His C, Fertin B, Bigan M, Dhulster P, Millares M, Froidevaux R (2022) Bioprocesses for the Biodiesel Production from Waste Oils and Valorization of Glycerol. Energies 15:3381. https://doi.org/https://doi.org/10.3390/en15093381
Dolejš I, Líšková M, Krasňan V, Markošová K, Rosenberg M, Lorenzini F, Marr AC, Rebroš M (2019) Production of 1, 3-propanediol from pure and crude glycerol using immobilized clostridium butyricum. Catalysts 9:317. https://doi.org/https://doi.org/10.3390/catal9040317
Drożdżyńska A, Pawlicka J, Kubiak P, Kośmider A, Pranke D, Olejnik-Schmidt A, Czaczyk K (2014) Conversion of glycerol to 1, 3-propanediol by Citrobacter freundii and Hafnia alvei–newly isolated strains from the Enterobacteriaceae. New biotechnology 31:402-410. https://doi.org/https://doi.org/10.1016/j.nbt.2014.04.002
Gonen C, Gungormusler M, Azbar N (2012) Comparative evaluation of pumice stone as an alternative immobilization material for 1, 3-propanediol production from waste glycerol by immobilized Klebsiella pneumoniae. Applied biochemistry and biotechnology 168:2136-2147. https://doi.org/https://doi.org/10.1007/s12010-012-9923-1
Gonen C, Gungormusler M, Azbar N (2013) Continuous production of 1, 3-propanediol using waste glycerol with Clostridium beijerinckii NRRL B-593 immobilized on glass beads and glass rushing rings. Chemical and Biochemical Engineering Quarterly 27:227-234.
Gungormusler-Yilmaz M, Shamshurin D, Grigoryan M, Taillefer M, Spicer V, Krokhin OV, Sparling R, Levin DB (2014) Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1, 3-propanediol synthesis at high glycerol loading. Amb Express 4:1-14.
Gungormusler-Yilmaz M, Cicek N, Levin DB, Azbar N (2016) Cell immobilization for microbial production of 1, 3-propanediol. Critical reviews in biotechnology 36:482-494. https://doi.org/https://doi.org/10.3109/07388551.2014.992386
Gungormusler M, Gonen C, Azbar N (2011) Use of ceramic‐based cell immobilization to produce 1, 3‐propanediol from biodiesel‐derived waste glycerol with Klebsiella pneumoniae. Journal of applied microbiology 111:1138-1147. https://doi.org/https://doi.org/10.1111/j.1365-2672.2011.05137.x
GÜNGÖRMÜŞLER M (2021) Production of value-added bioproducts using a modified continuous biofilm reactor by Citrobacter freundii DSM 15979. Hittite Journal of Science and Engineering 8:55-62. https://doi.org/http://dx.doi.org/10.17350/HJSE19030000213
Hazimah A, Ooi T, Salmiah A (2003) Recovery of glycerol and diglycerol from glycerol pitch. Journal of Oil Palm Research 15:1-5.
Hidayu A, Mohamad N, Matali S, Sharifah A (2013) Characterization of activated carbon prepared from oil palm empty fruit bunch using BET and FT-IR techniques. Procedia Engineering 68:379-384. https://doi.org/https://doi.org/10.1016/j.proeng.2013.12.195
Hodson ME (1998) Micropore surface area variation with grain size in unweathered alkali feldspars: Implications for surface roughness and dissolution studies. Geochimica et Cosmochimica Acta 62:3429-3435. https://doi.org/https://doi.org/10.1016/S0016-7037(98)00244-0
ISO (1976) Glycerines for industrial use - Determination of alkalinity or acidity - Titrimetric method. ISO 1615:1976 (E), International Organization for Standardization, Berlin, Germany
ISO (2010) Animal and vegetable fats and oils - Determination of ash. ISO 6884:2008(E), International Organization for Standardization, Switzerland
Jamali NS, Jahim JM, Isahak WNRW, Abdul PM (2017) Particle size variations of activated carbon on biofilm formation in thermophilic biohydrogen production from palm oil mill effluent. Energy Conversion and Management 141:354-366. https://doi.org/https://doi.org/10.1016/j.enconman.2016.09.067
Johnson DT, Taconi KA (2007) The glycerin glut: Options for the value‐added conversion of crude glycerol resulting from biodiesel production. Environmental Progress 26:338-348.
Jun S, Moon C, Kang C-H, Kong SW, Sang B-I, Um Y (2010) Microbial fed-batch production of 1, 3-propanediol using raw glycerol with suspended and immobilized Klebsiella pneumoniae. Applied biochemistry and biotechnology 161:491-501. https://doi.org/https://doi.org/10.1007/s12010-009-8839-x
Kaur G, Srivastava AK, Chand S (2012) Simple strategy of repeated batch cultivation for enhanced production of 1, 3-propanediol using Clostridium diolis. Applied biochemistry and biotechnology 167:1061-1068. https://doi.org/https://doi.org/10.1007/s12010-012-9715-7
Khanna S, Goyal A, Moholkar VS (2013) Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support. Biotechnology and Bioengineering 110:1637-1645. https://doi.org/https://doi.org/10.1002/bit.24839
Kim C, Song YE, Baek J, Im HS, Kim JR (2021) Zero-valent iron driven bioconversion of glycerol to 1, 3-propanediol using Klebsiella pneumoniae L17. Process Biochemistry 106:158-162. https://doi.org/https://doi.org/10.3390/fermentation9030233
Kowalska U, Mizielińska M (2017) Bioconversion of crude glycerol to 1, 3-propanediol by immobilized cells of Citrobacter freundii. World Scientific News 80:18-28.
Lammers P, Kerr B, Honeyman M, Stalder K, Dozier III W, Weber T, Kidd M, Bregendahl K (2008) Nitrogen-corrected apparent metabolizable energy value of crude glycerol for laying hens. Poultry science 87:104-107.
Lan Y, Feng J, Guo X, Fu H, Wang J (2021) Isolation and characterization of a newly identified Clostridium butyricum strain SCUT343-4 for 1, 3-propanediol production. Bioprocess and Biosystems Engineering 44:2375-2385. https://doi.org/https://doi.org/10.1007/s00449-021-02610-x
Laura M, Monica T, Dan-Cristian V (2020) The effect of crude glycerol impurities on 1, 3-propanediol biosynthesis by Klebsiella pneumoniae DSMZ 2026. Renewable Energy 153:1418-1427. https://doi.org/https://doi.org/10.1016/j.renene.2020.02.108
Lee C, Aroua MK, Daud WMAW, Cognet P, Pérès-Lucchese Y, Fabre P-L, Reynes O, Latapie L (2015) A review: conversion of bioglycerol into 1, 3-propanediol via biological and chemical method. Renewable and Sustainable Energy Reviews 42:963-972.
Lee K-C, Lim MSW, Hong Z-Y, Chong S, Tiong TJ, Pan G-T, Huang C-M (2021) Coconut shell-derived activated carbon for high-performance solid-state supercapacitors. Energies 14:4546. https://doi.org/https://doi.org/10.3390/en14154546
Loureiro‐Pinto M, González‐Benito G, Coca M, Lucas S, García‐Cubero MT (2016) Valorization of crude glycerol from the biodiesel industry to 1, 3‐propanediol by Clostridium butyricum DSM 10702: influence of pretreatment with ion exchange resins. The Canadian Journal of Chemical Engineering 94:1242-1248. https://doi.org/https://doi.org/10.1002/cjce.22501
Loureiro‐Pinto M, Coca M, González‐Benito G, Lucas S, García‐Cubero MT (2017) Continuous bioproduction of 1, 3‐propanediol from biodiesel raw glycerol: operation with free and immobilized cells of Clostridium butyricum DSM 10702. The Canadian Journal of Chemical Engineering 95:819-826. https://doi.org/https://doi.org/10.1002/cjce.22725
Luthfi AAI, Jahim JM, Harun S, Tan JP, Mohammad AW (2017) Potential use of coconut shell activated carbon as an immobilisation carrier for high conversion of succinic acid from oil palm frond hydrolysate. RSC advances 7:49480-49489. https://doi.org/https://doi.org/10.1039/C7RA09413B
Luthfi AAI, Tan JP, Harun S, Manaf SFA, Jahim JM (2019) Homogeneous solid dispersion (HSD) system for rapid and stable production of succinic acid from lignocellulosic hydrolysate. Bioprocess and biosystems engineering 42:117-130. https://doi.org/https://doi.org/10.1007/s00449-018-2019-8
Maina S, Kachrimanidou V, Ladakis D, Papanikolaou S, de Castro AM, Koutinas A (2019) Evaluation of 1, 3-propanediol production by two Citrobacter freundii strains using crude glycerol and soybean cake hydrolysate. Environmental Science and Pollution Research 26:35523-35532. https://doi.org/https://doi.org/10.1007/s11356-019-05485-4
Mehrotra T, Dev S, Banerjee A, Chatterjee A, Singh R, Aggarwal S (2021) Use of immobilized bacteria for environmental bioremediation: A review. Journal of Environmental Chemical Engineering 9:105920. https://doi.org/https://doi.org/10.1016/j.jece.2021.105920
Mizielińska M, Kowalska U, Sobol M (2020) The continuous bioconversion of glycerol to 1, 3-propanediol using immobilized Citrobacter. Biotechnol Lett 25:1448-1455. https://doi.org/http://dx.doi.org/10.25083/rbl/25.2/1448.1455
Moon C, Ahn J-H, Kim SW, Sang B-I, Um Y (2010) Effect of biodiesel-derived raw glycerol on 1, 3-propanediol production by different microorganisms. Applied biochemistry and biotechnology 161:502-510. https://doi.org/https://doi.org/10.1007/s12010-009-8859-6
Nanda M, Yuan Z, Qin W, Poirier M, Chunbao X (2014) Purification of crude glycerol using acidification: effects of acid types and product characterization. Austin J Chem Eng 1:1-7.
Nupueng S, Oosterveer P, Mol AP (2018) Implementing a palm oil‐based biodiesel policy: The case of Thailand. Energy Science & Engineering 6:643-657. https://doi.org/https://doi.org/10.1002/ese3.240
Rodriguez A, Wojtusik M, Ripoll V, Santos VE, Garcia-Ochoa F (2016) 1, 3-Propanediol production from glycerol with a novel biocatalyst Shimwellia blattae ATCC 33430: operational conditions and kinetics in batch cultivations. Bioresource technology 200:830-837. https://doi.org/https://doi.org/10.1016/j.biortech.2015.10.061
Samul D, Leja K, Grajek W (2014) Impurities of crude glycerol and their effect on metabolite production. Annals of microbiology 64:891-898. https://doi.org/https://doi.org/10.1007%2Fs13213-013-0767-x
Sittijunda S, Reungsang A (2020) Valorization of crude glycerol into hydrogen, 1, 3-propanediol, and ethanol in an up-flow anaerobic sludge blanket (UASB) reactor under thermophilic conditions. Renewable Energy 161:361-372. https://doi.org/https://doi.org/10.1016/j.renene.2020.07.053
Stegmann P (2014) The environmental performance of biobased 1, 3-propanediol production from glycerol compared to conventional production pathways-A Life Cycle Assessment.
Suratago T, Nootong K (2012) Production of 1, 3-propanediol by Clostridium butyricum DSM 5431 in an Anaerobic Moving-Bed Bioreactor. Macedonian Journal of Chemistry and Chemical Engineering 31:245–253-245–253. https://doi.org/https://doi.org/10.20450/mjcce.2012.23
Szymanowska-Powalowska D (2014) 1, 3-Propanediol production from crude glycerol by Clostridium butyricum DSP1 in repeated batch. Electronic Journal of Biotechnology 17:322-328. https://doi.org/https://doi.org/10.1016/j.ejbt.2014.10.001
Tan JP, Tee ZK, Roslam Wan Isahak WN, Kim BH, Asis AJ, Jahim JM (2018) Improved fermentability of pretreated glycerol enhanced bioconversion of 1, 3-propanediol. Industrial & Engineering Chemistry Research 57:12565-12573. https://doi.org/https://doi.org/10.1021/acs.iecr.8b02268
Tey KY, Tan JP, Yeap SK, He N, Bukhari NA, Hui YW, Luthfi AAI, Manaf SFA (2023) Current analysis on 1, 3-propanediol production from glycerol via pure wild strain fermentation. Journal of Environmental Chemical Engineering 110998. https://doi.org/https://doi.org/10.1016/j.jece.2023.110998
van Heerden C, Farzad S, Görgens JF (2023) Techno-Economic and Environmental Assessments of 1, 3-Propanediol and Xylooligosaccharide Production Annexed to a Typical Sugar Mill. ACS Sustainable Chemistry & Engineering 11:16453-16468.
Wang X-L, Zhou J-J, Sun Y-Q, Xiu Z-L (2019) Bioconversion of raw glycerol from waste cooking-oil-based biodiesel production to 1, 3-propanediol and lactate by a microbial consortium. Frontiers in bioengineering and biotechnology 7:14. https://doi.org/https://doi.org/10.3389/fbioe.2019.00014
Wang X-L, Zhou J-J, Shen J-T, Zheng Y-F, Sun Y-q, Xiu Z-L (2020) Sequential fed-batch fermentation of 1, 3-propanediol from glycerol by Clostridium butyricum DL07. Applied Microbiology and Biotechnology 104:9179-9191. https://doi.org/https://doi.org/10.1007/s00253-020-10931-2
Wilkens E, Ringel AK, Hortig D, Willke T, Vorlop K-D (2012) High-level production of 1, 3-propanediol from crude glycerol by Clostridium butyricum AKR102a. Applied microbiology and biotechnology 93:1057-1063. https://doi.org/https://doi.org/10.1007/s00253-011-3595-6
Wirawan SS, Solikhah MD, Setiapraja H, Sugiyono A (2024) Biodiesel implementation in Indonesia: Experiences and future perspectives. Renewable and Sustainable Energy Reviews 189:113911. https://doi.org/https://doi.org/10.1016/j.rser.2023.113911
Woo WX, Koh HS, Tan JP, Yeap SK, Abdul PM, Luthfi AAI, Manaf SFA (2022) An overview on cell and enzyme immobilization for enhanced biohydrogen production from lignocellulosic biomass. International Journal of Hydrogen Energy 47:40714-40730. https://doi.org/https://doi.org/10.1016/j.ijhydene.2022.08.164
Wu J, Wang F, Wang Z, Ye H, Liu P (2016) 1, 3-Propanediol production from crude glycerol by Klebsiella pneumoniae. Chiang Mai J Sci 43:718-725.
Yang X, Kim DS, Choi HS, Kim CK, Thapa LP, Park C, Kim SW (2017) Repeated batch production of 1, 3-propanediol from biodiesel derived waste glycerol by Klebsiella pneumoniae. Chemical Engineering Journal 314:660-669. https://doi.org/https://doi.org/10.1016/j.cej.2016.12.029
Yang X, Choi HS, Lee JH, Lee SK, Han SO, Park C, Kim SW (2018) Improved production of 1, 3-propanediol from biodiesel-derived crude glycerol by Klebsiella pneumoniae in fed-batch fermentation. Chemical Engineering Journal 349:25-36. https://doi.org/https://doi.org/10.1016/j.cej.2018.05.042
Yen H-W, Li F-T, Wong C-L, Chang J-S (2014) The pH effects on the distribution of 1, 3-propanediol and 2, 3-butanediol produced simultaneously by using an isolated indigenous Klebsiella sp. Ana-WS5. Bioprocess and biosystems engineering 37:425-431. https://doi.org/https://doi.org/10.1007/s00449-013-1008-1
Zabed HM, Zhang Y, Guo Q, Yun J, Yang M, Zhang G, Qi X (2019) Co-biosynthesis of 3-hydroxypropionic acid and 1, 3-propanediol by a newly isolated Lactobacillus reuteri strain during whole cell biotransformation of glycerol. Journal of Cleaner Production 226:432-442. https://doi.org/https://doi.org/10.1016/j.jclepro.2019.04.071
Zareba JK, Nyk M, Samoc M (2016) Co/ZIF-8 heterometallic nanoparticles: control of nanocrystal size and properties by a mixed-metal approach. Crystal Growth & Design 16:6419-6425. https://doi.org/https://doi.org/10.1021/acs.cgd.6b01090
Zdarta J, Meyer AS, Jesionowski T, Pinelo M (2019) Multi-faceted strategy based on enzyme immobilization with reactant adsorption and membrane technology for biocatalytic removal of pollutants: a critical review. Biotechnology advances 37:107401. https://doi.org/https://doi.org/10.1016/j.biotechadv.2019.05.007
Zhang AH, Liu HL, Huang SY, Fu YS, Fang BS (2018) Metabolic profiles analysis of 1, 3‐propanediol production process by Clostridium butyricum through repeated batch fermentation coupled with activated carbon adsorption. Biotechnology and bioengineering 115:684-693. https://doi.org/https://doi.org/10.1002/bit.26488
Zhang H, Hwang S, Wang M, Feng Z, Karakalos S, Luo L, Qiao Z, Xie X, Wang C, Su D (2017) Single atomic iron catalysts for oxygen reduction in acidic media: particle size control and thermal activation. Journal of the American Chemical Society 139:14143-14149. https://doi.org/https://doi.org/10.1021/jacs.7b06514
Zhou W, Liu J, Fan S, Xiao Z, Qiu B, Wang Y, Li J, Liu Y (2018) Biofilm immobilization of Clostridium acetobutylicum on particulate carriers for acetone-butanol-ethanol (ABE) production. Bioresource Technology Reports 3:211-217. https://doi.org/https://doi.org/10.1016/j.biteb.2018.08.008
Zulqarnain, Yusoff MHM, Ayoub M, Jusoh N, Abdullah AZ (2020) The challenges of a biodiesel implementation program in Malaysia. Processes 8:1244. https://doi.org/https://doi.org/10.3390/pr8101244

Competing interest reported. The guest editor, Dr. Woon Kok Sin is involved in this research and is one of the coauthors.

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Fermentation of acidic-pretreated glycerol for enhanced 1,3-PDO production by immobilized Clostridium butyricum JKT 37 on coconut shell activated carbon

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Acid pretreatment of crude glycerol

2.2 Characterization of glycerol samples

2.3 Preparation of microbial inoculum and fermentation media

2.4 Cell density improvement in fermentation using different mesh sizes of CSAC as immobilizers

2.5 Batch fermentation of suspended and immobilized cell

2.6 Reusability study of the immobilized cell fermentation at an optimum initial substrate concentration

2.7 Analytical methods

3. RESULTS AND DISCUSSION

3.1 Characterization of glycerol samples

3.2 Cell density improvement by immobilized C. butyricum JKT 37

3.4 Surface morphology visualization of immobilized C. butyricum JKT 37 on CSAC

3.5 Fermentation of pure glycerol fermentation by suspended and immobilized C. butyricum JKT 37

3.6 Fermentation of different glycerol by suspended and immobilized C. butyricum JKT 37

3.7 Determination of optimum 1,3-PDO yield from different initial substrate concentration

3.8 Reusability study of the immobilized C. butyricum JKT 37

CONCLUSION

Abbreviations

Declarations

References

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Supplementary Files

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