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Article

Sustainable Biofuel Production from Sludge by Oleaginous Fungi: Effect of Process Variables on Lipid Accumulation

by
Habib Ullah
1,2,†,
Muzammil Anjum
3,*,†,
Bushra Noor
3,
Samia Qadeer
4,
Rab Nawaz
5,
Azeem Khalid
3,
Aansa Rukaya Saleem
6,
Bilal Kabeer
7,
Abubakr M. Idris
8,9,
Muhammad Tayyab Sohail
10,11,12 and
Zepeng Rao
1,2,*
1
Department of Environmental Science, Zhejiang University, Hangzhou 330058, China
2
Innovation Center of Yangtze River Delta, Zhejiang University, Jiashan 311400, China
3
Department of Environment Sciences, Pir Mehr Ali Shah Arid Agriculture University, Shamsabad, Rawalpindi 46300, Pakistan
4
Department of Environmental Sciences, Allama Iqbal Open University, Islamabad 44310, Pakistan
5
Centre for Tropical Climate Change System, Institute of Climate Change, Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
6
Department of Earth and Environmental Sciences, Bahira University, Islamabad 44000, Pakistan
7
Imam Turki bin Abdullah Royal Nature Reserve Development Authority, Riyadh 12511, Saudi Arabia
8
Department of Chemistry, College of Science, King Khalid University, Abha 62529, Saudi Arabia
9
Research Center for Advanced Materials Science (RCAMS), King Khalid University, Abha 62529, Saudi Arabia
10
Centre for Sustainable Development, Gulf University for Science and Technology, Hawally 32093, Kuwait
11
Applied Science Research Center, Applied Science Private University, Amman 11931, Jordan
12
Department of Public Administration, School of Business, The University of Jordan, Amman 11942, Jordan
 
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*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Catalysts 2025, 15(11), 1009; https://doi.org/10.3390/catal15111009
Submission received: 10 July 2025 / Revised: 12 September 2025 / Accepted: 14 September 2025 / Published: 27 October 2025
(This article belongs to the Special Issue Catalysis Accelerating Energy and Environmental Sustainability)
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Abstract

The current paper investigated the potential of oleaginous fungus Rhizopus oryzae B97 for lipid accumulation under varying process variables. The fungal strain was isolated from bread mold and analyzed for its potential to grow on sludge with simultaneous production of microbial lipids. The sludge sample was sourced from the wastewater treatment plant located in Sector I-9, Islamabad. The effects of various process variables, such as pH, temperature, carbon and nitrogen sources, and shaking, on lipid accumulation, cell dry weight (CDW), chemical oxygen demand (COD), and volatile solids (VS) removal were investigated. It was found that glucose and yeast promoted the maximum lipid accumulation. At the same time, the fungal biomass reached its maximum value of up to 64% at 30 °C and at pH 4 (CDW: 28 g/L). These process conditions also improved the sludge treatment efficiency, achieving 68% COD and 55% VS removal in 168 h. FTIR analysis of the accumulated lipids indicated strong characteristic peaks of functional groups associated with fatty acids. The GC-MS analysis confirmed the production of essential FAMEs required in biodiesel production from the corresponding fatty acids, such as oleic acid, palmitic acid, stearic acid, and erucic acid. Operation in a continuous-shaking aerobic batch reactor (CSABR) system under optimum conditions further improved the process efficiency. Overall, the results indicated the competent potential of oleaginous fungus Rhizopus oryzae B97 for lipid-based biofuel production through fatty acid transesterification.

1. Introduction

Pakistan, which was once a water-surplus country is now heading towards water shortage in terms of availability. The main reason behind this loss is wastewater, either industrial or municipal, which remains untreated and directly flows into water bodies and sewer systems [1]. According to one estimate, the level of solid waste generation in Pakistan is 50,000 tons per day, out of which only 60% is collected and then disposed of properly [2]. The rapid increase in the human population and their enhanced living standards cannot meet the current energy demand. Three-quarters of energy production is carried out through the consumption of fossil fuels, which is the main cause of the release of carbon dioxide into the atmosphere, which will ultimately result in air pollution as well as climate change [3]. Energy plays vital role in the economy of Pakistan. Pakistan is a net importer of energy because the majority of its energy supply is generated by fossil fuels such as oil, coal, and gas, as well as hydro and nuclear power. For its energy production, Pakistan is dependent on hydrocarbon fuels, the consumption of which results in excessive CO2 emissions and ultimately leads to climate change [4]. Although the current level of emissions in Pakistan is low, the path towards sustainability can only be achieved by adopting better energy practices.
Concern about the global energy and fuel production crisis is gaining importance, and alternative methods of fuel production are continuously being explored. To combat the energy crisis in Pakistan, renewable energy resources can be used as the best alternative source of energy in terms of wind energy, solar energy, and biomass-based energy. Biomass-based bioenergy is gaining attention for its potential in combating energy shortages [5]. The challenges associated with using these alternative sources of energy are feedstock availability cost, land requirements, and water supply. Despite some challenges faced by previous generations of biofuels, fourth-generation biofuel is now considered the best alternative, which involves the use of genetically modified algae, fungi, and bacteria [6]. Biofuels that are generally produced are cleaner than fossil fuels, as they are usually carbon-neutral and produce relatively lower levels of toxic emissions upon burning. These biofuels include biogas, bioethanol, biodiesel, and green fuels, which are all types of cleaner biofuels [7]. Advanced technologies used for converting waste into bioenergy include biochemical processes (anaerobic digestion, transesterification, and fermentation) and thermochemical processes (pyrolysis, liquefaction, and gasification). Out of these, transesterification is the simplest and most economical technology [8].
The fungal treatment of sludge uses less energy. The oxygen requirements of fungi are roughly one-third of the oxygen requirements of a bacterial population. Furthermore, the fungi utilize all supplies available to optimize the decomposition of organic matter. Fungi have an advantage over bacteria because they can break down a wider range of complex and diverse substrates [9]. Furthermore, these microorganisms are also well known for their ability to produce oil and are recognized as natural oil producers [10]. It was reported by an estimate that the lipid accumulation potential of various fungi, algae, and yeasts is 70–90% higher than that of bacteria at 20–50% [11]. On earth, there are about 1.5–5.0 million fungal species [12]. These fungi typically possess an enzymatic system that enables them to efficiently break down lignocellulosic materials and other complex molecules. Some of the fungal species reported as natural oil producers include Mortierella isabellina, Aspergillus flavus, and Mucor circinelloides, among others [13].
Sewage sludge production is pertinent in wastewater treatment plants, and depending on the type of treatment process applied and type of wastewater, the sludge can have varying characteristics [14]. The quality and quantity of sewage sludge are controlled by various factors, including the type of water, pollutant load, type and concentration of industrial effluent, treatment technology, and sludge parameters, such as the age of the sludge, chemicals used, and sludge stabilization methods [15]. Studies have highlighted the interest of researchers in the resource recovery and recycling possibilities of sludge during the sludge management process [16]. However, generally, sludge is 99% water and contains different concentrations of biosolids, other organic compounds, and micro-pollutants if advanced methods of treatment are not applied [17]. In previous studies, the significance of biological, chemical, thermal, and mechanical pretreatments has also been highlighted, offering the disintegration of sludge flocks and thus providing a better substrate [18,19]. These characteristics make sludge a promising candidate for resource recovery through the use of heterotrophic organisms.
In previous studies, different types of waste have been processed by filamentous fungi for lipid accumulation and simultaneous waste treatment. For instance, Zheng and colleagues [20] studied 11 filamentous fungi strains, and a lipid content of 39.4% was obtained from the Mortierella isabellina strain. It was also demonstrated in their research that this approach is a viable alternative to oil and can deliver excellent lipid production. In another study, oleaginous filamentous fungi, Cunninghamella echinulate, were cultivated on orange peel and glucose, and a lipid content of 46.6% and a 14.1% linolenic acid content were obtained [10]. Filamentous fungi can be used to produce oils by using substrates such as glycerol, wheat straw, and acetic acid [21]. Similar studies have also reported lipid accumulation from fungi using industrial waste [22] and agro-industrial waste [23]. Researchers have also demonstrated lipid accumulation using wastewater sludge [24,25]. Furthermore, the potential of fungi to produce cleaner fuels has also been presented in previous research [26].
To convert these lipids stored in the cell biomass, transesterification is usually performed [27]. Transesterification is a process for converting oil and lipids extracted from plants into esters and glycerol in the presence of a catalyst and alcohol [8]. Several controlling factors can affect the transesterification process, including temperature, reaction time, type of alcohol, mode of reaction conditions, molar ratio, type of catalyst, and purity of the reactants [28]. The chemical structure (chain length and number of double bonds) of the fatty acids present in the lipid feedstock has a considerable influence on the fuel and quality parameters of the biodiesel produced [29]. Biofuel production from stored lipids is also dependent on the type of FAMEs produced [27], where the majority of the fungal species are capable of synthesizing FAMEs ranging from C16:0–C18:0, which are ideal for biofuel production due to their close resemblance to plant-based biofuels [30]. Therefore, these fungal strains can be employed for lipid accumulation and corresponding biofuel production. The accumulation of lipids for the production of oils and biofuels is a relatively novel approach. It is best achieved through the transesterification method, which utilizes strains of filamentous fungi to capture lipids in their cell biomass. This process is one of the most sustainable and eco-friendly methods for producing biofuels in huge quantities [31]. Keeping in mind the characteristics of sewage sludge and the potential of filamentous fungi, the current study aimed to harness sludge from a municipal wastewater treatment plant and use it for potential biolipid accumulation using fungi and for the conversion of stored lipids into biofuel through transesterification.

2. Results and Discussions

2.1. Physico-Chemical Characterisation of Wastewater Sludge

The physico-chemical characterization of the collected sludge samples is illustrated in Table 1. The pH of the wastewater sludge was 6.64, and the temperature was 20 °C, both of which are optimal for microbial growth. The volatile solids content of 3.4% indicates a significant concentration of organic material. A high COD value up to 6400 mg/L and a total dissolved solids content of 815 mg/L indicated the presence of a significant organic load and level of nutrients. Sludge usually contains high COD values [32,33], and Reda et al. [34] found a COD value of up to 25,842 mg/L in thickened sludge. Notably, the exceptionally high carbon content (52.52%) with a low nitrogen content (0.19%) indicates a high C/N ratio, which could be suitable for the growth of oleaginous fungi and lipid accumulation, highlighting the potential of sludge as a biodiesel feedstock.
To determine the metal content in the collected waste sample, ICP-OES was used, and the results are listed below in Table 2. It was observed that both toxic and essential elements were detected in varying concentrations in the sludge. The highest level (21.21 mg/L) was found for K. Among the heavy metals, Cd showed a significantly higher concentration, i.e., 16.73 mg/L, and Cu and Co were detected at moderate levels, i.e., 0.72 mg/L and 0.2 mg/L, respectively. Very low concentrations of As, Cr, Ni, and Pb were detected at very low concentrations, ranging from 0.01–0.02 mg/L. The existence of heavy metals may be attributed to the fact that the wastewater treatment plant receives not only sewage and domestic water but also wastewater from industries located in the vicinity. Moreover, approximately up to 80 % of heavy metals (such as Cd, Cu, Pb, Zn, Ni, Cr, Mn, and Hg) in sewage enter it through biological and physico-chemical interactions during the sewage treatment process [35,36]. Overall, the data suggest that cadmium is the primary pollutant of concern in this sample, while other metals remain at comparatively lower levels.

2.2. Isolation of Oleagenous Fungi

Initially, the fungal strains were isolated based on different-colored colonies that appeared on culture media plates. A total of 13 different strains were isolated from all sources based on their morphological characteristics. Thereafter, purification of the strains was performed by cultivating the isolated strains on petri plates several times until a pure and single colony was observed on each plate. Based on pure growth, only seven strains (B97, P40, C33 W19, S21, R25, and SR42) were finally isolated for further processing.
For the identification of fungi at the genus level, macroscopic and microscopic examinations were performed based on their shape, size, color, margins, and elevation, as reported by Alsohaili and Bani-Hasan [37]. Table 3 presents the morphological characteristics and possible genera of the seven isolated fungal strains. The strains isolated from bread (B97 and P40) possibly belonged to the genera Rhizopus and Mucor, respectively. Strains C33 (citrus)and SR42 (sweets), isolated from citrus, may belong to Aspergilus or Penicillium. The morphological characteristics of S21 (soil) and R25 (rocks) showed a close resemblance to Mucor.

2.3. Screening of Oleagenous Fungi

After pure growth was observed, primary screening was then performed using liquid CDB media. It was observed that all seven isolated strains showed excellent growth, and a lipid accumulation potential of more than 20% when cultivated in CBD media. In a secondary screening, the strains were cultivated in liquid sludge and analyzed for their lipid accumulation in CDW.
The lipid accumulation potential of the seven isolated fungal strains cultivated on sludge is illustrated in Figure 1, which shows a significant difference (p = 1.61 × 10−8) among all strains. In the case of the control, no lipid accumulation was observed, which revealed that sludge alone does contribute to lipid production in the absence of a fungal strain. B97 showed the highest lipid accumulation of up to 56% after 168 h, confirming its strong oleaginous potential. Following this, strains W19 and P40 also achieved significantly higher lipid accumulation potential (45% and 44%, respectively). The lowest lipid yield was shown by the C33 strain isolated from citrus, which suggested its weak lipid production capacity. These observations are consistent with previous studies, where lipid accumulation by different fungal strains was observed to range from 20 to 80% [38,39]. For instance, the fungal strain M. circinelloides showed a lipid accumulation level of up to 60% using corn steep solids as a feedstock [40]. Similarly, in another study, Mucor circinelloides achieved a lipid accumulation level of up to 43% when cultivated using glucose as a carbon source [41]. Therefore, the high lipid production by B97 (Rhizopus oryzae) underscores its potential as a promising fungus for use in sustainable biofuel generation from sludge.

2.4. Effect of Different Process Variables

The selected Rhizopus oryzae B97 fungal strain was further investigated to analyze the effect of various operating factors on its lipid accumulation potential and its ability to treat sludge. These factors included pH, temperature, carbon, nitrogen, and shaking conditions.

2.4.1. Effect on Lipid Accumulation Potential

The results show that the lipid accumulation by Rhizopus oryzae B97 varied significantly (p < 0.05) due to the influence of varying operating conditions (Figure 2). In the case of pH, the highest lipid accumulation (54%) was achieved at an acidic pH range (4) after 168 h (Figure 2A). In the case of temperature, lipid accumulation reached 58% at 30 °C (Figure 2B), which indicates that moderate or lower mesophilic conditions are more suitable compared to significantly low or high extremes. Similar findings were reported by Papanikolaou and Aggelis [42], who achieved the maximum lipid accumulation at mesophilic temperatures in Mucor and Rhizopus species. Furthermore, it has been reported that high temperatures produce lower levels of unsaturated fatty acids compared to saturated fatty acids, where incubation at low temperature is generally effective for the production of unsaturated fatty acids [43]. The lipid accumulation was further enhanced by varying the shaking conditions, where at 150 rpm, a 59% level of lipid accumulation was observed (Figure 2E), which was possibly due to the improved nutrient availability and oxygen transfer. Appropriate shaking conditions provide constant turbulence, which is needed for increasing biomass production and lipid accumulation [44]. The use of nutrient supplements, i.e., carbon and nitrogen, showed a significant impact when using glucose and yeast extract, respectively, and this achieved the highest production, i.e., up to 64% (Figure 2C,D), demonstrating their supportive role in improving lipid synthesis in Rhizopus oryzae.

2.4.2. Cell Dry Weight

The growth of Rizopous oryae (B97) in terms of CDW was influenced by various factors, as illustrated in Figure 3A. A significant statistical difference (p > 0.05) was observed among all the varying operating conditions. The maximum CDW of 21 g/L was achieved at pH 4 (Figure 3A), which was also observed in the case of lipid accumulation, confirming that acidic pH is favorable for fungal growth. The mesophilic temperature of 30 °C achieved the highest CDW (21 g/L) (Figure 3B), corresponding to the highest level of lipid accumulation at the same temperature. Figure 3C shows that CDW was influenced differently by the different carbon sources; for glucose, the maximum CDW was 25 g/L, followed by lactose (19 g/L) and dextrose (15 g/L), thus again confirming glucose to be the most suitable supplement as a carbon source. The use of yeast extract as a nitrogen source (Figure 3D) achieved the highest fungal growth, i.e., up to 28 g/l (CDW). Nitrogen sources are considered an essential part of microbial growth substrates that can significantly influence fungal growth and the subsequent accumulation of lipids and unsaturated fatty acids [45]. Overall, these findings reveal that the isolated fungal strains (Rhizophus oryzae B97) grow best at pH 4 and at a temperature of 30 °C with supplementation of glucose and yeast extract as carbon and nitrogen sources, respectively.

2.4.3. COD Removal

The removal of organic matter from the sludge under different operating conditions was monitored as a factor of COD removal and VS removal. The effect of pH indicated a significant change in COD across different time intervals (Figure 4). Interestingly, Rhizopus oryzae B97 exhibited the highest degradation at a pH range of 4.0, followed by pH 9, pH 8, pH 6, pH 7, and pH 5. The change in degradation was highest at pH 4 (55%) and lowest at pH 5 (48%), which can be attributed to the inherent capacity of the fungus to offer better degradation in acidic environments. However, the strain Rhizopus oryzae B97 is rich in hydrolytic enzymes, which offer better degradation in acidic environments. Considering the current scenario, degradation was also observed at neutral and basic pH levels, which can be linked to possible enzymatic activities at these pH levels [46]. Another reason for the high level of degradation under acidic conditions may be the increased solubility of organic compounds under acidic conditions [47]. At pH 4, the maximum COD removal was observed at 30 °C, which was significantly higher than that at other temperatures. Ideally, hydrolytic enzymes work best under a temperature range of 37–40 °C. This inconsistent degradation pattern could be linked to different factors, including better oxygen provision at 30 °C, which can decline with a rise in temperature [48], or less thermal activation of the enzymes.
The three different carbon sources tested for COD removal indicated that the addition of a carbon source significantly improved the COD removal efficiency by the strain Rhizopus oryzae B97, raising the maximum COD removal from 58% (Figure 4B) to 66% (Figure 4C). The maximum degradation was observed with the addition of glucose, while the other two sources lagged behind, with removals of 61% and 56% obtained with the addition of dextrose and lactose, respectively. Similarly, (Figure 4D), the addition of nitrogen also increased the COD removal. Contrary to the addition of a carbon source, the addition of nitrogen sources demonstrated interesting and competitive potential with all three nitrogen sources, offering a 68% COD removal with yeast, a 65% removal with sodium nitrate, and a 66% removal with ammonium chloride. However, due to its better economic value and slightly better performance, yeast can be considered as an ideal nitrogen supplement. Following the enhancement in degradation through the addition of nutrients, the rate of COD removal was also monitored at different rpm levels, ranging from 50 rpm to 150 rpm. The effect of a change in rpm on degradation was also examined by Boutafda et al. [49]. However, we found that the maximum degradation was obtained by reducing the rpm to 150–50, which resulted in a decline in COD removal efficacy from 54% to 45% (Figure 4E). Interestingly, this removal percentage was lower than the previous one (Figure 4D).

2.4.4. VS Removal

The degradation of organic matter was further confirmed by monitoring the removal of VS. Like the COD removal results, the maximum VS removal was also achieved at pH 4, but interestingly, subsequent degradation was achieved at pH 8, pH 6, pH 9, and pH 5, while the minimum VS removal, i.e., 28%, was achieved at pH 7 (Figure 5A). Thus, this indicates that an acidic pH might be favorable for the removal of oxidizable organics, but the trend for the reduction in bulk solids was minimal at neutral pH. Following the investigation of the effects of pH, the effect of temperature variation were also monitored. The maximum VS removal was also achieved at 30 °C (41%), and overall, competent removal was achieved (Figure 5B). The addition of different carbon sources also demonstrated the same effect; due to it being a simpler source of sugar, the addition of glucose considerably improved the VS removal level to 46% (Figure 5C). The addition of other carbon sources also considerably increased the VS removal level. This can be attributed to the fact that better nutrient availability enhanced fungal growth and, in turn, VS removal. The degradation was further enhanced by the addition of a nitrogen source (Figure 5D), indicating 55% VS removal in 168 h with the addition of yeast extract, followed by 50% with ammonium chloride and 53% with ammonium chloride. However, changing the rpm did not deliver very promising results, as a reduction in rpm to 150 rpm removed 42% of the VS, and the maximum VS removal was achieved at 100 rpm, which was 43%.

2.5. Continuous-Shaking Aerobic Batch Reactor (CSABR) Performance

A CSABR experiment was conducted to analyze the potential of the isolated B97 strain (Rhizopus oryzae) for simultaneous lipid-based biofuel production and sludge treatment. A bioreactor is a biologically active tank that provides an ideal environment for a biological agent to degrade a substrate [50]. It was observed that the reactor demonstrated a significant (p = 0.0032) improvement in lipid accumulation and sludge treatment performance (Figure 6A). The lipid accumulation percentage increased over time and reached 68% after 7 d (168 h), whereas in the control reactor (without a fungal strain), only 2% lipid accumulation was observed. This was due to the use of isolated Rhizopus oryzae fungus that has been reported as an efficient oleaginous species among various fungi [51]. Similarly, the fungal biomass (CDW) level reached 16 g/L after 168 h, while no fungal biomass was observed in the control reactor (Figure 6B). The efficiency of the sludge treatment was measured by observing the effect on COD and VS removal. Figure 6C shows that the COD removal efficiency was substantially improved from 22% (24 h) to 79% (168 h), demonstrating the efficient degradation of organic matter in the sludge. The treatment efficiency of the present study is significantly higher than that of a previous study conducted by Zhu and coworkers [52], where up to 45% COD removal in sludge was achieved with Phanerochaete chrysosporium fungi. In the case of VS, a similar trend was observed, where up to 64% VS removal was achieved after 168 h. This indicates that Rhizopus oryzae B97 has an excellent ability to degrade the organic matter present in the sludge. Overall, the CSABR system under controlled optimum operating conditions demonstrated the efficient growth of Rhizopus oryzae B97 fungi, lipid accumulation, and simultaneous sludge treatment, leading to the resource recovery.

2.6. Lipid Profile Analysis Through FTIR Spectroscopy

FTIR spectroscopy is a non-destructive method that can be used to analyze the total biochemical profile of both intercellular and extracellular metabolites of fungal cells [53]. The lipid profile of the extracted lipids from the B97 fungal strains was examined using FTIR spectroscopy in the spectral range of 400–4000 cm−1, as illustrated in Figure 7. A set of characteristic peaks were observed for various organic compounds, including lipids, in the spectral regions of 3000–2850 cm−1, 1700–1300 cm−1, 1100–1050 cm−1, and 880–600 cm−1. A close, similar lipid detection range was also reported by Forfang et al. [54]. The lipids can be identified by peaks related to aliphatic C-H stretching vibrations, such as C-H stretching in –CH3 at 2888.69 cm−1, CH2 at 2970.6 cm−1, and C-O-C stretching in esters at 1054.84 cm−1 and 1084.65 cm−1, as lipids typically show aliphatic C-H stretching vibrations. These ranges closely correspond to the reported values of Shurvell [55] and Maquelin et al. [56]. The absorbance values at 1654.93 cm−1 represent the presence of significant amounts of free fatty acids in the extracted fungal lipids. The absorbance peak at 1379.12 cm−1 indicates the –CH3 of acyl chains in fatty acids. These acyl chains could be associated with fatty acids of triacylglycerols (TAGs) [57], which are known as the primary storage lipids of fungal cells. Similarly, the minor peaks at 805.10 cm−1 and 615.01 cm−1 (CH2) are associated with acyl chains in fatty acids of TAGs. The other signals ranging from 2950–2900 cm−1 (a) may be related to glucuronic acid and glycerol.

2.7. Biodiesel Production (Transesterification of Extracted Lipids)

The GC-MS analysis of the transesterified lipids extracted from the isolated fungal strain B97 confirmed the production of various FAMEs, showing the potential of the fungal strain for biodiesel production (Table 4). The predominant component found was 9-octadecenoic acid methyl ester (C18:1) [58], which accounted for the most significant proportion by area, i.e., 38.69%. This indicates that MUFAs predominated in the lipid profile. Furthermore, significant concentrations of 13-docosenoic acid methyl ester (C23:2), with an area of 9.00%, and erucic acid methyl ester (C22:1), with a proportion of 5.71% were detected, contributing to the fraction of MUFAs. Furthermore, the detection of oleic acid (C18:0) is considered to be ideal for biodiesel production because it provides balanced conditions between oxidative stability and cold flow characteristics [59].
In case of saturated fatty acids, hexadecanoic acid methyl ester (C16:0) and methyl stearate (C18:0) were predominant, with a proportional area of 7.67% and 1.22%. The presence of SFAs in biodiesel generally improves the cetane number [60] and oxidative stability; however, excessive levels may lead to a negative impact on cold flow properties. The presence of polyunsaturated fatty acids, such as 11,14-eicosadienoic acid methyl ester (0.41%; C20:2) and linoleic acid ethyl ester (1.43%; C18:2), proposes that the lipids produced by the fungal strain B97 contain essential fatty acid components, albeit in lower concentrations than MUFAs.
Overall, the GC-MS results showed that fungal strain B97 grown on sludge can produce a significant amount of MUFAs, mainly oleic acid, which is regarded as an optimal fatty acid for biodiesel feedstock due to its excellent fuel balance [61]. These outcomes are consistent with previous research that reported oleic-acid-rich fungal strains could be used as a potential biodiesel resource [62] The transesterified mixture of saturated and unsaturated fatty acids suggests that biodiesel produced from the B97 strain would likely have high oxidative stability and low-temperature characteristics, making it an ideal renewable fuel.

3. Methodology

3.1. Sampling of Wastewater Sludge and Physico-Chemical Characterization

The sludge samples were collected from the wastewater treatment plant located in Sector I-9, Islamabad, Pakistan (33.650993° N, 73.053219° E). This treatment plant receives both sewage/domestic wastewater as well as industrial effluent from industries located in the industrial zones of Islamabad city. The samples were collected in sterilized bottles and preserved in an ice box. The samples were transported to the ENV research lab of PMAS-Arid Agriculture University, Rawalpindi, Pakistan, and stored in cold storage before physico-chemical characterization.
Physico-chemical characterization of the sludge samples was performed following the American Public Health Association (APHA) standard protocols for various parameters [63], i.e., pH, EC, TDS, TSS, VS, FS, TS, moisture content, COD, total nitrogen, and metal analysis by ICP-OES.

3.2. Strain Isolation

Fungal isolation was carried out from different sources (soil, bread mold, sweets, apple, rocks, and citrus). From each source, the fungal cells were isolated on agar plates. The isolated strains were further enriched in potato dextrose agar (PDA) (Thermofisher Scientific, Waltham, MA, USA) media at pH 7. To inhibit bacterial growth, Spectinomycin was added to the PDA and incubated at 28 °C for 7 days [64]. Thereafter, the isolated strains were purified by cultivating multiple times on PDA media plates.
After purification of the fungal strains, initial screening of the potential oleaginous strains was conducted on the basis of lipid accumulation in CDW. The isolated strains were cultured in 200 mL of Czapek Dox Broth (CDB) (Millipore Sigma, Darmstadt, Germany) along with a control. Fungal strains demonstrating more than 20% lipid accumulation were subject to a secondary screening process. Secondary screening consisted of inoculating the sludge (250 mL; sterilized) with 5 mL of the inoculum of each fungal strain in separate flasks. The lipid accumulation in the fungal CDW was measured over a time period ranging from 24 h–168 h at different time intervals. These strains were labeled as S21, R25, SR24, B97, C33, P40, and W19 and were identified based on detailed morphological characterization [37].

3.3. Experimental Setup

Fungal strain B97, which showed the highest lipid accumulation potential, was selected for further experimentation. A set of experimental setups were conducted, each containing 250 mL of sludge with 5% (v/v) inoculum. All experiments were set for 7 days, and readings were taken after 24 h, 96 h, 120 h, 144 h, and 168 h. The samples were subjected to physico-chemical estimation, including lipid yield, cell dry weight (CDW), optical density (OD), pH, electrical conductivity (EC), total dissolved solids (TDS), chemical oxygen demand (COD), and volatile solids (VS). The effect of different parameters, such as temperature, pH, incubation time, shaking condition, and carbon and nitrogen sources, was monitored. The factor-wise optimization included four different temperatures of 25 °C, 30 °C, 35 °C, and 40 °C followed by optimization of pH at 4, 5, 6, 7, 8, and 9 and then shaking conditions at 50 rpm, 100 rpm, and 150 rpm, Under the best-suited conditions, lipid accumulation was further optimized in terms of carbon sources, including glucose, lactose, and dextrose, at a sample concentration of 5 g/L. For the optimized C:N ratio, the experiment was conducted with three nitrogen sources, i.e., yeast, ammonium chloride, and sodium nitrate, which were applied at a concentration of 0.5 g/L of the sample. The best-suited conditions were further used in the continuous-shaking aerobic batch reactor (CSABR).

3.4. Continuous-Shaking Aerobic Batch Reactor (CSABR)

This experiment consisted of a bioreactor with a 250 mL reaction volume containing 250 mL of sludge and a 5% v/v inoculation of the fungal strain (v/v). The operational parameters were fixed as per those obtained in the previous section, i.e., pH 4, temperature of 30 °C, and glucose and yeast as carbon and nitrogen sources, respectively, with shaking at 150 rpm. The reaction volume and conditions were maintained for 7 days, and the final output in terms of lipid accumulation and COD removal was monitored.

3.5. Transesterification of Extracted Lipids

The extracted lipids from the CASBR were subject to acid hydrolysis for transesterification. Briefly, transesterification was performed by mixing 1 mL of chloroform and 1 mL of methanol into the lipids (10:1 v/v), with 15% H2SO4 used as a catalyst [28]. The mixture was heated in a water bath for 2.5 h, and a distinct layer of biofuel was formed. The biofuel thus formed was transferred to a vial for FTIR analysis.

3.6. Analytical Procedures

The waste degradation parameter analyses were performed using the APHA standard methods [63]. For instance, total nitrogen, COD, and VS were estimated using 4500- APHA-2005, +5220-APHA-2005, and 5240-APHA-2005, respectively. TDS, EC, and a pH were monitored using an HI-98301 TDS meter (Hanna Instruments, Inc., Woonsocket, RI, USA), an EC meter (Milwaukee EC40, Rocky Mount, NC, USA), and a PHS-3C pH meter (Hangzhou Qiwei Instrument Co., Ltd., Hangzhou, China), respectively. Metal analysis was carried out by using the ICP-OES standard method. A brief description of other parameters is provided below.

3.6.1. Lipid Content

The lipid content was measured via gravimetric analysis using a modified version of the Bligh and Dyer method, as reported by Briel et al. [65]. For the estimation of the lipid content in the cell biomass, the obtained cell dry weight was further extracted using a using standard chloroform/methanol extraction method. The lyophilized pellets were initially mixed with a 2:1 (v/v) chloroform–methanol ratio. The formula used for lipid % is given below:
L i p i d   % = W e i g h t   o f   E x t r a c t e d   L i p i d   ( g ) C e l l   D r y   W e i g h t   ( g ) × 100

3.6.2. Lipid Analysis by FTIR Analysis

Previously, FTIR has been reported to show lipid accumulation from oil-producing microbes [54]. Therefore, FTIR of the extracted lipid was performed to analyze the lipid profile, i.e., possible fatty acids present in the lipid content. The analysis was performed using the micro-lab method from FTIR Agilent technology, with a range of 4000–400.

3.6.3. Post-Transesterified Lipid Analysis (FAMEs) by GC-MS

For the FAME analysis of the extracted lipid content, initial GC-MS analysis was conducted following the same protocol discussed in our previous research [66]. Briefly, the extracted samples were transesterified (as discussed in Section 2.6), and subject to further analysis, the esterified lipids were filtered through a syringe filter containing Na2SO4 with a pore size of 0.2 micron, thus separating all solids and moisture under anoxic conditions. The details of the GC-MS conditions are given in Akbar et al. [66].

4. Conclusions

In the present study, seven fungal strains were isolated based on their best growth potential. Among these, the strains isolated from bread (B97 and P40) exhibited the highest growth and lipid accumulation. Notably, the fungal isolate B97 (Rhizopus oryzae) achieved the highest lipid accumulation, confirming its strong oleaginous potential. The most suitable conditions for Rhizopus oryzae B97 were determined to be pH 4, a temperature of 30 °C, shaking at 150 rpm, and glucose and yeast extract used as carbon and nitrogen sources, respectively. Under these process conditions, the lipid accumulation reached its maximum level (64%), while the fungal CDW-based biomass was 28 g/L. The sludge treatment efficiency was also improved, achieving 68% COD and 55% VS removal in 168 h, indicating the potential of Rhizopus oryzae B97 for organic removal. The operation in the CSABR system under optimum conditions further improved the process efficiency and control over fungal growth, sludge treatment, and lipid-based biofuel production. In the future, scaling-up this process using Rhizopus oryzae B97 fungi could contribute to sustainable biofuel production and effective sludge treatment.

Author Contributions

Conceptualization, methodology, investigation, formal analysis, writing—original draft: H.U.; supervision, project administration, funding acquisition, writing—review and editing: M.A.; methodology, investigation, formal analysis, writing—original draft: B.N.; validation, resources, formal analysis: S.Q.; writing—review and editing: R.N.; validation, writing—review and editing: M.T.S.: validation, writing—review and editing: B.K.; writing—review and editing: A.M.I.; writing—review and editing: A.K.; writing—review and editing: A.R.S.; supervision, funding acquisition, writing—review and editing: Z.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Key Research and Development Program of Zhejiang Province, China (2024C03125) and National Natura Science Foundtion of China (22506180). Part of this work is supported under grant number NCPC/Cb/F40-1/30012025 by the National Cleaner Production Center Foundation. The authors also extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through General Research Project under grant number GRP/130/45.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Catalysts 15 01009 g001
Figure 1. Lipid accumulation percentage in isolated fungal strains.
Figure 1. Lipid accumulation percentage in isolated fungal strains.
Catalysts 15 01009 g001
Catalysts 15 01009 g002
Figure 2. Lipid accumulation potential of Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Figure 2. Lipid accumulation potential of Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Catalysts 15 01009 g002
Catalysts 15 01009 g003
Figure 3. CDW of Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Figure 3. CDW of Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Catalysts 15 01009 g003
Catalysts 15 01009 g004
Figure 4. COD removal in sludge using Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources), (D) effect of nitrogen sources, (E) effect of shaking conditions.
Figure 4. COD removal in sludge using Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources), (D) effect of nitrogen sources, (E) effect of shaking conditions.
Catalysts 15 01009 g004
Catalysts 15 01009 g005
Figure 5. Volatile solids removal in sludge using Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Figure 5. Volatile solids removal in sludge using Rhizopus oryzae B97; (A) effect of pH, (B) effect of temperature, (C) effect of carbon sources, (D) effect of nitrogen sources, (E) effect of shaking conditions.
Catalysts 15 01009 g005
Catalysts 15 01009 g006
Figure 6. Bioreactor performance using Rhizopus oryzae B97 under optimized conditions: (A) lipid accumulation potential %, (B) cell dry weight, (C) COD removal %, (D) VS removal %.
Figure 6. Bioreactor performance using Rhizopus oryzae B97 under optimized conditions: (A) lipid accumulation potential %, (B) cell dry weight, (C) COD removal %, (D) VS removal %.
Catalysts 15 01009 g006
Catalysts 15 01009 g007
Figure 7. FTIR spectrum of extracted lipids from Rhizopus oryzae B97 strain. In the figure, “a” represents 2950–2900 cm−1.
Figure 7. FTIR spectrum of extracted lipids from Rhizopus oryzae B97 strain. In the figure, “a” represents 2950–2900 cm−1.
Catalysts 15 01009 g007
Table 1. Physico-chemical characteristics of wastewater sludge.
Table 1. Physico-chemical characteristics of wastewater sludge.
S. No.Liquid SludgeResults
1.Total dissolved solids (mg/L)815
2.Total suspended solids (mg/L)300
3.Total solids %3.5
4.Fixed solids %0.1
5.Volatile solids %3.4
6.COD (mg/L)6400
7.Total nitrogen %0.19
8.Total carbon %52.52
9.pH6.64
10.EC (µs/cm)700
11.ColorLight brownish-grey that turns brown over time
12.Moisture %97
Table 2. Metal analysis of wastewater sludge.
Table 2. Metal analysis of wastewater sludge.
S. No.MetalConcentration (mg/L)
1.Arsenic (As)0.01
2.Cadmium (Cd)16.73
3.Cobalt (Co)0.2
4.Chromium (Cr)0.01
5.Copper (Cu)0.72
6.Potassium (K)21.21
7.Nikel (Ni)0.01
8.Lead (Pb)0.02
Table 3. Morphological characteristics of the selected fungal strains.
Table 3. Morphological characteristics of the selected fungal strains.
SourceLabelFormColorElevationMarginPossible Genera
BreadB97Filamentous
Irregular
Rhizoid		White, brown, black/greyUmbonate
Convex
Fluffy		Diffused/spreading outwardsRhizopus (Rhizopus oryzae)
BreadP40Filamentous
Irregular
Rhizoid		White, green, brown, blackUmbonate
Convex		Filiform
Entire		Mucor
CitrusC33Filamentous
Irregular
Circular		White, black, brown, greenRaised
Convex		Filiform
Lobate
Undulate		Aspergillus/Penicillium
AppleW19IrregularWhite, brownFlat
Convex		FiliformAlternaria/Cladosporium
SoilS21FilamentousGreenRaisedFiliformMucor
RocksR25Filamentous
Rhizoid		GreenRaisedFiliformRhizopus/Mucor
SweetsSR42Filamentous
Irregular
Rhizoid		Dark green, whiteConvexFiliformAspergillus/Penicillium
Table 4. FAME profile of transesterified lipids with their corresponding lipids produced by Rhizopus oryzae B97.
Table 4. FAME profile of transesterified lipids with their corresponding lipids produced by Rhizopus oryzae B97.
Area %FAMS (Esters) FormedPossible Corresponding LipidNo of Carbon Atoms: Double BondCompound Category
0.22Methyl tetradecanoateMyristic acidC14:0SFA
7.67Hexadecanoic acid, methyl esterPalmitic acidC16:0SFA
Pentadecanoic acid, 14-methyl, methyl ester14-Methylpentadecanoic acidC15:0SFA (branched)
0.11Cis-10-heptadecenoic acid, methyl esterCis-10-heptadecenoic acidC17:1MUFA
38.699-Octadecenoic acid, methyl esterOleic acidC18:1MUFA
1.22Methyl stearateStearic acidC19:0SFA
4.99Oleic acidOleic acidC18:0MUFA
1.43Linoleic acid ethyl ester-C18:2PUFA
1.63Ethyl oleate-C20:2UFA
0.4111,14-Eicosadienoic acid, methyl esterArachidic acidC20:2PUFA
9.0013-docosenoic acid, methyl ester C23:2MUFA
5.71Erucic acidErucic acidC22:1MUFA
0.63Tetracosanoic acid, methyl ester C24:0SFA
SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty acid, UFA: unsaturated fatty acid.
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MDPI and ACS Style

Ullah, H.; Anjum, M.; Noor, B.; Qadeer, S.; Nawaz, R.; Khalid, A.; Saleem, A.R.; Kabeer, B.; Idris, A.M.; Sohail, M.T.; et al. Sustainable Biofuel Production from Sludge by Oleaginous Fungi: Effect of Process Variables on Lipid Accumulation. Catalysts 2025, 15, 1009. https://doi.org/10.3390/catal15111009

AMA Style

Ullah H, Anjum M, Noor B, Qadeer S, Nawaz R, Khalid A, Saleem AR, Kabeer B, Idris AM, Sohail MT, et al. Sustainable Biofuel Production from Sludge by Oleaginous Fungi: Effect of Process Variables on Lipid Accumulation. Catalysts. 2025; 15(11):1009. https://doi.org/10.3390/catal15111009

Chicago/Turabian Style

Ullah, Habib, Muzammil Anjum, Bushra Noor, Samia Qadeer, Rab Nawaz, Azeem Khalid, Aansa Rukaya Saleem, Bilal Kabeer, Abubakr M. Idris, Muhammad Tayyab Sohail, and et al. 2025. "Sustainable Biofuel Production from Sludge by Oleaginous Fungi: Effect of Process Variables on Lipid Accumulation" Catalysts 15, no. 11: 1009. https://doi.org/10.3390/catal15111009

APA Style

Ullah, H., Anjum, M., Noor, B., Qadeer, S., Nawaz, R., Khalid, A., Saleem, A. R., Kabeer, B., Idris, A. M., Sohail, M. T., & Rao, Z. (2025). Sustainable Biofuel Production from Sludge by Oleaginous Fungi: Effect of Process Variables on Lipid Accumulation. Catalysts, 15(11), 1009. https://doi.org/10.3390/catal15111009

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