Introduction

The most pressing environmental issue facing urban and rural areas in many African nations is solid waste management. Improper handling of these agricultural wastes has posed a significant challenge over the past decades. The African continent is responsible for approximately 10–15 million metric tonnes of solid waste from agro-industrial activities1,2. Cassava processing industries are the main contributors to this waste, with effluents from cassava processing often discharged untreated3. Cassava, scientifically known as Manihot esculenta Crantz, is revered for its versatility, resilience in diverse climates, and crucial role in food security for millions in tropical regions4. Over 700 million people in tropical regions rely on its starchy storage roots as a primary energy source5. Cassava is a reliable and affordable source of carbohydrates for people in Sub-Saharan Africa, particularly in Nigeria, where its production, processing, and consumption are most predominant and significant on a global scale6. However, Kolawole3 noted that cassava by-products are increasingly contributing to global hazardous waste, industrial disasters, and environmental health risks. Hence, there is a critical need for cost-effective and tailored solutions to achieve sustainable waste minimisation in response to the growing demand for waste-free environments and the challenges of costly waste management7.

The increasing number of cassava processing industries in most tropical countries has given rise to considerable quantities of waste and residues as these companies expand their capacity with new factories for added value products. These wastes include peels from cassava initial processing, fibrous by-products from the crushing and sieving of tuber, starch residues together with cassava bagasse arising during processing, as well as huge volumes of wastewater effluents8,9. The costs of processing and treating these wastes for disposal tend to swallow a significant portion of the profits made by cassava processing industries, especially in rural areas/countries that are still heavily agrarian. As a result, rural cassava processors often dispose of these waste products into the environment and water bodies without treatment during processing, leading to significant alterations in the receiving aquatic ecosystems10. This pollutes water and soil and, in return, can have a direct or indirect impact on human health. Therefore, sustainable and low-cost waste management approaches are needed to tackle this environmental challenge as well as secure the future sustainability of cassava processing industries in rural areas.

The utilisation of bio-procedures in waste management and effluent treatment is beneficial as it not only prevents adverse environmental effects from direct disposal but also transforms these wastes into valuable by-products, offering a sustainable solution8,11,12. Bioprocesses can transform cassava waste into starch, citric acids, biofuels, animal feed, biodegradable plastics, and more. These bio-procedures, like anaerobic digestion and composting, are biological processes in nature and hence might further reduce the negative effects of cassava waste on the environment. In the cassava processing industry, this technique will support sustainable waste management and reduce pollution impacting nature. It will also yield environmental and economic benefits from other valuable byproducts.

Hence, this study aimed to adequately demonstrate biotechnologies in the exploitation and conversion of cassava peels and effluents into useful products. The effectiveness of cassava effluent treatment in reducing Biological Oxygen Demand (BOD5) and cyanide concentration was analysed. The potential of biofiltration methods for removing pollutants from cassava wastewater before discharge was evaluated. Enzymatic separation of cassava starch from peels and residues and citric acid production from cassava peels using A. niger as a microbial agent were also investigated. The filtration was done to remove coarse solids and fibres from cassava processing effluent. Wastewater is passed through the sand, gravel, and charcoal filters to reduce turbidity and separate suspended solids. Enzymatic treatment with cellulase and pectinase was used to get starch back from the peels and solid waste. Fermentation with A. niger was then used to turn the glucose-rich hydrolysate into citric acid. The enzymatic treatment helped break down the cellulose and pectin in the peels, making it easier to extract the starch. Fermentation with A. niger then converted the glucose into citric acid, a valuable product for various industries. This application of bio-procedures was expected to offer sustainable solutions for managing these by-products and recover more value from materials previously viewed as waste.

Materials and methods

Experimental site

The study area was Oyo State, Nigeria. A locale situated in the derived savanna vegetation belt of Nigeria, which is used for cultivation. Cassava is one of the major crops grown in this state. It is a staple food for many people in the region due to its versatility and nutritional value13. Four major medium-size processing hubs were chosen. Adekunle Fajuyi Cantonment (AFC) in Odogbo-Ojoo, Ibadan; Letmauck Cantonment (LMC) in Mokola, Ibadan; Onipepeye (OIR) on Old Ife Road, Ibadan; and Ile Ileri (IIO) in Odo-Oba, Ogbomoso, based on their massive processing volumes and output.

Methods

Sample collection and Preparation

Cassava effluent was collected from processing centres and stored in 50-litre polypropylene containers. These cassava effluents were treated with an integrated physical treatment system composed of anaerobic and aerobic processes for the treatment of primary effluent as outlined by Selvamurugan, et al.14 to reduce pollution. Thereafter, cassava effluent samples were divided into 4 portions (25 l each). The first portion was incubated for 24 h, the second portion for 48 h, the third portion for 72 h, and the fourth portion was left without incubation. After treatment, 10 l of the effluent from each sample were collected into sterile rubber containers kept under refrigeration at 20 °C before usage.

Filtration procedure

The charcoal utilised was hard charcoal sourced from a charcoal depot and had undergone the pyrolysis process for preparation. This entails combusting hardwood at elevated temperatures in an oxygen-deprived environment. The charcoal was boiled to eliminate surface contaminants and to expand the minuscule holes between the carbon atoms. The material was subsequently dried in a hot air oven (Model: TO008GA-34, AKAI-TOKOYO, Japan) at 45 °C for 10 h. The desiccated charcoal was pulverised with a mortar and pestle into minute fragments and subsequently sieved into three distinct particle sizes: 2 mm for coarse, 0.5 mm for granular, and 0.1 mm for fine charcoal. The sand and gravel were also washed, dried and classified into three distinct particle sizes: 2 mm for coarse, 0.5 mm for granular, and 0.1 mm for fine. The filters were improvised and consist of a 2-litre plastic bottle with the bottom section removed. Small apertures were created in the bottle cap, and a fine mesh material was employed to seal the smaller opening, so preventing the filtration media (sand, gravel, and charcoal) from escaping or contaminating the filtrate. The filter is a multi-layer bed composed of different-sized particles of the same material. Each filter column was filled from bottom to top with 20 mm of fine (198 g), 80 mm of medium (764 g), and 40 mm of coarse (382 g) filtering materials (Fig. 1). The filters were rinsed with distilled water before the filtration of the treated samples. The prepared cassava effluent sample was gradually introduced into the filter and permitted to percolate through the filtration layers. Following the completion of the filtering process, the sample was reintroduced three times to achieve three filtration cycles. Each filtration cycle processed 1.5 l of cassava effluent within 30 to 45 min, yielding a flow rate of 33 to 50 mL/min. The calculated contact duration with the filter medium was 20 to 25 min, facilitating adequate interaction for the elimination of suspended particles and adsorptive purification. The filtered sample was then examined.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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A schematic diagram of the filtration process.

Cassava effluent treatment for biological oxygen demand [BOD5] and cyanide determination

The initial concentrations of total cyanide and BOD5 in the raw effluent were determined by following methods developed by Guédé, et al.15 for cyanide, and the APHA16 standard procedure was selected to determine BOD5. Thereafter, and the filtrates were then retested for the levels of BOD5 and cyanide contents.

Enzymatic separation of cassava starch from peels and residues

Starch was recovered from peels and solid residues through a process that included cellulolytic and pectinolytic enzymes, as described by Kallabinski and Balagopalan17with some modifications. Cellulase (Celluclast 1.5 l from Trichoderma reesei) and Pectinase from A. niger were purchased from Sigma-Aldrich, a subsidiary of Merck KGaA, Darmstadt, Germany. Fresh cassava peels obtained from each processing site were washed and air-dried at ambient temperature for two days. Subsequently, 100 g of each was ground using a Kenwood Power Blender (Model: Kenwood Power Blender + Mill 1000 W BLM45.240SS). The ground mash was allocated to 250 ml conical flasks, each containing 50 ml of water. Enzyme dosages were applied at concentrations of 0.2%, 0.3%, and 0.4% (w/w) based on the dry weight of cassava peel. Treatments included cellulase-only, pectinase-only, and a combined treatment (1:1 weight ratio of cellulase and pectinase). A control experiment devoid of enzyme treatment was established to compare the amount of recovered starch. The enzyme-treated mixtures were incubated for 2 to 3 h at 30 °C under static conditions, with intermittent stirring. The isolated starch suspension was permitted to settle overnight. The supernatant was removed, and the settled starch was recovered by filtration through 80-mesh sieves, yielding a pure product for subsequent analysis. The experiment was conducted three times, and the mean result was reported. The starch samples acquired after enzymatic separation underwent pasting analysis.

The pasting profiles of the recovered starch samples were analysed using a Rapid Visco-Analyser (RVA) from Newport Scientific Pty. Ltd. with Thermocline for Windows version 1.1 software (1996). The RVA was connected to a PC monitor for real-time recording of the pasting properties and curves. A starch suspension was prepared by adding 3.0 g of dry starch to distilled water to achieve a total weight of 28.0 g in the RVA sample canister. The paddle was placed inside the canister, centrally aligned with the paddle coupling, and then inserted into the RVA machine. The measurement cycle was initiated by pressing the motor tower of the instrument. The 12-minute profile was displayed on the connected computer monitor. The time-temperature regime of the equipment was as follows: it started at 50 °C for 1 min, then heated to 95 °C over 3 min, held at 95 °C for 3 min, and then cooled back to 50 °C over 4 min. Finally, the temperature was maintained at 50 °C for 1 min. The equivalent sample weight (S) and volume of water (W) were calculated using Eqs. 1 and 2.

$$\:\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{S}\right)=\frac{A\times\:100}{100-M}$$
(1)
$$\:\text{V}\text{o}\text{l}\text{u}\text{m}\text{e}\:\text{o}\text{f}\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\left(\text{W}\right)=28-\text{S}$$
(2)

Where; A = 3 g, M = moisture content of the sample, W = volume of water, 28 g is the total weight of the suspension in the RVA canister, and S = calculated sample weight for RVA.

The following parameters were measured in RVA units: peak viscosity (the highest viscosity during the 95 °C heating stage), holding strength (the lowest viscosity at the end of the 95 °C heating stage), breakdown (the change in viscosity from peak to holding strength), cold paste or final viscosity (the highest viscosity at the end of the 50 °C cooling stage), and setback (the change in viscosity from holding strength to final viscosity).

Citric acid production from cassava peels using A. niger

Citric acid was extracted from cassava peels following the process described by Ajala, et al.18 and Ajala, et al.19with minor modifications. Figure 2 outlines the citric acid production processes. The production of citric acid involves fermentation of sugars by microorganisms such as A. niger. The final product is then purified and concentrated for use in various industries such as food and beverage, pharmaceuticals, and cosmetics. Cassava peels sourced from the processing centres were milled and sieved to obtain particles sized 0.8–2.0 mm. Five (5 mL) of distilled water were added to 25 g of each sample, which were then autoclaved at 121 °C for 20 min to gelatinize the residual starch. Thereafter, these treated cassava peel samples were supplemented with a salt solution containing urea (2.93 g/L), KH2PO4 (1.86 g/L), and FeSO4·7H2O (0.0105 g/L), previously autoclaved at 121 °C for 15 min.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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The citric acid production processes.

At the Central Research Laboratory of Ladoke Akintola University of Technology, Ogbomoso, a fully-grown A. niger (LAU 09) culture was collected and sub-cultured for 7 weeks to obtain pure microbial cultures on potato dextrose agar. These cultures were incubated at 32 °C for 72 h and stored on agar slants at 4 °C until needed. The treated substrate was inoculated with spores (10^8 spores/g of treated cassava peel), and methanol, in the range of 0–4% v/w, was added under sterile conditions. The culture media were incubated at 25–37 °C for 72 h, with the contents of each flask stirred 3–4 times a day to ensure uniform microbial action throughout the medium. The effects of methanol concentration and temperature on the fungal production of citric acid from cassava peel were evaluated. Citric acid production in each sample was determined using a titrimetric method with 0.1 M NaOH and phenolphthalein as an indicator.

Statistical analysis

All experiments were conducted in triplicate, and data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way Analysis of Variance (ANOVA) to evaluate the significance of differences among treatment means. To find out if the differences between the means were significant, Duncan’s multiple range test was used, and a p-value less than 0.05 was judged significant. All experimental computations and graph plotting were done using Microsoft Excel spreadsheet software version 2016.

Results and discussion

BOD5 in effluent filtered through sand, gravel and charcoal medium

Table 1 shows the biochemical oxygen demand (BOD5) (mg/L) in cassava effluent filtered through sand, gravel, and charcoal medium at different incubation periods. The BOD5 range through sand medium for all the processing centres was 1041–1271 (24 h), 972–1169 (48 h), and 875–1031 (72 h) compared with the control 2467–3631 (0 h). The BOD5 range through gravel medium for all the processing centres was 993–1405 (24 h), 718–1104 (48 h), and 670–910 (72 h) compared with the control 2467–3631 (0 h). On the other hand, the BOD5 range through charcoal medium for all the processing centres was 873–1000 (24 h), 610–805 (48 h), and 393–620 (72 h) compared with the control 2467–3631 (0 h).

Sand is the most common filter media in effluents treatment, and it has a relatively small specific surface area (0.136 m2/g) and a high bulk density of 1690 kg/m3,20,21. In the current study, the purifying efficacy of sand was evaluated against that of gravel and charcoal, both of which possess lower bulk densities and a greater specific surface area than sand. The application of all filtration media significantly (p < 0.05) decreased the BOD5 levels in cassava effluents across all processing centres. The use of charcoal medium resulted in the lowest BOD5 levels (393–825 mg/L) over the 72-hour incubation period in comparison to alternative filter media. This implies that charcoal can be a potential filtration bed to lower down BOD5 concentration in cassava effluents compared to sand and gravel. Future work could investigate the possibility of integrating various filtration materials to enhance purification efficiency.

The BOD5 of the effluent significantly decreased after 72 h of incubation with charcoal, gravel, and sand. The reductions for charcoal were 84% (AFC) and 77% (IIO); for gravel, 81% (LMC) and 63% (AFC); and for sand, 74% (LMC) and 65% (AFC). The extensive specific surface area of charcoal facilitated elevated BOD5 removal rates (55–84%), while gravel filters exhibited intermediate to high removal rates (44–81%), and sand filters demonstrated the lowest rates (51–74%). This showed that charcoal filters were more effective in removing BOD5 from the effluent Dalahmeh, Pell, Vinnerås, Hylander, Öborn and Jönsson21 also reported similar findings that the bark and charcoal filters were more effective in eliminating contaminants from graywater due to their extensive surface area, facilitating the absorption and mineralisation of organic materials by microorganisms.

On the other hand, due to sand’s lower specific surface area and low porosity, less removal of organic matter was achieved21,22. Assiddieq, et al.23 and Ibaid24 earlier stated that the mass of filtering medium has a great impact with respect to time in which organic substances get absorbed, which also provided validation towards the same goal here. Adsorbent masses that are greater absorb more organic materials, reducing the BOD5 within the wastewater. The shape and size of the filtering medium itself play a substantial role in how well it removes pollutants from graywater. Studies indicate that media with varying sizes, such as irregularly shaped media, offer more surface area for adsorption than uniform sand particles23,24.

Even though the BOD₅ levels were lowered a lot, the final concentration is still higher than the WHO (industrial discharge to surface waters) limit of ≤ 50 mg/L and the EPA (secondary treatment standard) limit of ≤ 30 mg/L12. This means that more post-treatment, like aerobic polishing or constructed wetlands, may be needed before the water can be safely released. It is essential to contemplate the implementation of additional treatment processes to guarantee adherence to regulatory standards and safeguard aquatic environments from potential damage. Employing these modern treatment techniques can facilitate the requisite decrease in BOD₅ levels for safe release into surface waters.

Table 1 BOD5 (mg/L) in effluent filtered through sand, gravel and charcoal medium at different incubation periods.
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Total HCN in effluent incubated and filtered through sand, gravel and charcoal media

Table 2 presents the total HCN concentration (mg/L) in the effluent following a 24-hour incubation and three cycles of filtering through sand, gravel, and charcoal medium. HCN concentrations in effluent filtered through sand varied from 1.04 to 2.13 mg/L (24 h), 0.98 to 2.01 mg/L (48 h), and 0.94 to 1.83 mg/L (72 h), in contrast to control levels of 3.63 to 4.27 mg/L (0 h). For gravel filtration, HCN levels were 0.99 to 2.33 mg/L (24 h), 0.72 to 2.10 mg/L (48 h), and 0.67 to 1.91 mg/L (72 h) compared to the control. For charcoal filtration, HCN levels ranged from 0.87 to 1.64 mg/L (24 h), 0.80 to 1.01 mg/L (48 h), and 0.43 to 0.83 mg/L (72 h) compared to the control. Significant differences (p < 0.05) were observed in all tested samples from various locations. However, the activated carbon column treatment was the most effective at getting rid of HCN. This shows that activated charcoal is better at reducing the organic load than the sand filter.

The initial HCN concentrations in the effluents, ranging from 3.63 mg/L to 4.27 mg/L, are significantly above permissible limits, posing serious risks to aquatic ecosystems and human health if discharged without treatment. These concentrations align with findings from other studies, which have identified industrial effluents as a significant source of cyanide pollution in water bodies. After filtration, particularly with charcoal, HCN levels drop substantially. However, the final concentrations in some samples (e.g., AFC with 0.43 mg/L) still exceed the strictest guidelines for safe environmental discharge, such as those set by the EU (0.005 mg/L), EPA (0.2 mg/L), and WHO (0.07 mg/L)25,26. This indicates that while the filtration process is effective, it might need to be supplemented with additional treatment steps or dilutions to meet environmental safety standards.

The HCN concentrations after treatment, especially those below 1.0 mg/L, are less likely to cause acute toxicity to aquatic life. However, chronic exposure, even at low levels, can have long-term ecological impacts. For instance, fish and invertebrates are particularly sensitive to cyanide, with lethal concentrations (LC50) for some species being as low as 0.05 mg/L. The treated effluents, particularly those filtered through charcoal, show significantly lower HCN levels, which may be closer to or below permissible limits for environmental discharge. Therefore, effluents with HCN levels above 0.2 mg/L after treatment should undergo further cyanide-targeted detoxification steps such as alkaline chlorination and bioremediation using cyanide-degrading microbes before being discharged to ensure they comply with environmental safety standards.

Starch content of enzyme-treated cassava peel samples

The starch extracted from enzyme-treated cassava peel samples at a 5 ml dosage and a temperature of 45 oC for a period of 2 and 3 h is presented in Fig. 3. For the cassava peel sample collected from AFC, the yield significantly increased from 2.50 g at 2 h to 4.84 g at 3 h. This indicates that cellulase effectively breaks down cellulose over time, releasing more starch as hydrolysis progresses. A similar trend was seen with pectinase, where starch yield increased from 2.23 g to 3.81 g between 2 and 3 h. Pectinase mainly breaks down pectin in plant cell walls, aiding in the release of starch granules. The combination of cellulase and pectinase resulted in the highest starch extraction (5.30 g at 3 h), suggesting a synergistic effect. This combination likely leads to a more complete breakdown of cell wall components, releasing more starch.

Table 2 Total HCN (mg/L) in effluent incubated and filtered through sand, gravel and charcoal media.
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Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Starch content from enzyme-treated cassava peel samples. a – f: Mean values with different alphabets in the same enzymatic extraction are significantly different at p < 0.05. Where: AFC = Cassava peels from Adekunle Fajuyi Cantonment Processing Centre, Odogbo, Ibadan. LMC = Cassava peels Lekmauck Cantonment Processing Centre, Mokola, Ibadan. OIR = Cassava peels from Old Ife Road Processing Centre, Ibadan. IIO = Cassava peels from Ile Ileri Processing Centre, Odo Oba, Ogbomoso.

For the cassava peel sample collected from LMC, the yield increased from 2.45 g at 2 h to 4.95 g at 3 h, similar to the AFC sample. This consistency across different samples suggests that cellulase is effective in hydrolysing cellulose to release starch. The yield increased with pectinase treatment but remained lower than with cellulase (from 2.05 g to 2.45 g), indicating that pectinase alone may not be as effective in releasing starch. The combination treatment again resulted in the highest yield (5.12 g at 3 h), reinforcing the idea of a synergistic effect between these enzymes.

For the cassava peel sample collected from OIR, the starch yield increased from 2.86 g to 3.28 g over time. This sample showed a less dramatic increase in starch extraction compared to AFC and LMC, suggesting possible variability in cell wall composition or structure. The starch yield with pectinase treatment remained relatively low (1.94 g at 2 h to 2.01 g at 3 h), possibly due to the lower pectin content in this sample. The combination of cellulase and pectinase increased the yield from 2.95 g to 3.45 g, indicating the combined enzyme treatment’s effectiveness, though still lower than in AFC and LMC samples.

The cassava peel sample obtained from IIO had a significantly higher yield (3.74 g), which slightly increased to 3.93 g at both time points. This suggests that this sample may have high initial starch or cellulase compared with cassava peels collected from other centres. Also, the extraction yield seemed quite higher for pectinase, increasing from 3.57 to 3.65 g at both processing times. This increase is demonstrating proper pectin hydrolysis. The combined treatment had an increase from 3.63 g to 3.85 g, although this increase was less pronounced relative to other samples collected from AFC and LMC at 3 h processing time. The diversity in starch recovery among processing centres, especially the superior yield from AFC and LMC, can be ascribed to various biological and agronomic factors, including discrepancies in cassava species, maturity stage at harvest, and peel-to-flesh ratio. The presence of high-starch cultivars, proper harvest time, and effective processing likely enhanced enzymatic recovery27,28. These findings underscore the significance of upstream farming methods and varietal selection in optimising resource recovery from cassava waste streams. Adopting optimal procedures in cassava production and choosing high-starch cultivars can markedly increase starch recovery rates during processing, hence improving overall resource utilisation. This underscores the necessity for ongoing research and development to optimise agricultural methods for maximising the effectiveness of cassava waste use.

These enzymes have been used on a large scale to pretreat plant materials, which can be subsequently extracted following conventional extraction methods. Most of the time enzymes such as cellulases, pectinases, and hemicellulases are required to disrupt plant cell wall structure, leading to a greater extraction of bioactive compounds within plants. These enzymes led to a significant hydrolysis of cell wall constituents, which increased the permeability of their cell walls and hence yielded a high extraction yield for bioactives29. The complementary roles of cellulase and pectinase enhanced the breakdown efficiency of cassava fibrous leftovers. Cellulase functions by hydrolyzing β−1,4-glycosidic bonds in cellulose, effectively breaking down cellulose fibers into smaller sugar units such as cellobiose and glucose30,31. This action loosens the structural rigidity of the cell wall but may not be sufficient on its own due to the continued presence of intact pectin components. Pectinase, on the other hand, targets the pectin layer specifically the galacturonic acid backbone by cleaving α−1,4 linkages32,33. The application of pectinase weakens the integrity of the cell wall and increases the permeability of the substrate by breaking these intercellular connections. The synergy happens when pectinase gets the peel tissue ready by breaking down the pectic substances. This lets cellulase get deeper into the layers that are high in cellulose. This synergistic process markedly enhanced starch extraction from intricate plant tissues, underscoring the efficacy of multi-enzyme approaches in waste valorisation.

This enzymatic valorisation approach aligns with current trends in sustainable agro-waste bioprocessing and biofiltration technologies, which emphasise the use of low-energy, enzyme-mediated systems to convert agricultural residues into valuable bioproducts34,35. The synergistic application of cellulase and pectinase has also been shown to improve recovery efficiency and reduce processing costs in bioconversion and industrial fermentation settings. Also, researchers have found that using cellulase and pectinase together can greatly increase the amount of starch that is produced by targeting different parts of the cell wall. Zhang, et al.36 documented a significant enhancement in starch yield when these enzymes were utilised in conjunction, ascribing the advancement to the degradation of cellulose and pectin matrices around starch granules.

The period of treatment significantly impacted starch yield. Application for a shorter time of either cellulase or pectinase alone did not improve the extraction yield significantly from the peel. Compared to the control sample that was treated for two hours, 11.2%, 9.5%, and 12.2% of the total starch that was originally in the peel were recovered when treated with cellulase, pectinase, and a mix of the two in the conditions used in this study. The prolonged application of combination cellulase and pectinase for three hours yielded superior starch results. The starch yield was dependent upon the concentration of each enzyme and exhibited significant differences (p < 0.05) among treatments. The higher contents of starch yield over time support the results by Hameed, et al.37who demonstrated that more time of enzymatic treatment results in a complete hydrolysis or digestion for the cell wall components, and then an enhanced starch release was achieved. This study highlighted that prolonged enzyme exposure enables a more comprehensive breakdown, aligning with the tendencies identified in this research. However, some studies reported that differences in cell wall constitution and structure, as reported by Ortega, et al.38 and Cornejo, et al.39. can enhance enzyme efficacy among samples. Cornejo, Maldonado-Alvarado, Palacios-Ponce, Hugo and Rosell39 reported that the lignocellulosic composition of cassava varieties or plant parts could be different in a certain ratio to generate variable enzyme efficiencies with respect to starch recovery or yield. This justifies the increase in starch recovery efficiency without loss of quality by enzymatic treatment.

Therefore, enzyme treatment prior to extraction increases the yield of cassava starch without deteriorating the quality. The cellulase and pectinase treatment not only increases starch liberation from the peel but also improves susceptibility to α-amylase hydrolysation of additional remaining starchy materials. Starch granules in untreated peel are entrapped within a fibrous matrix, which limits their accessibility. This fibrous matrix is dehulled by celllase and pectinase, thereby exposing the starch granules to α-amylases40. Thus, this study revealed that the application of enzymatic treatment significantly enhanced starch extractability from cassava peels, making it a profitable option for starch production. In addition, using enzymes in this process is environmentally friendly and sustainable, which lessens the need for harsh chemicals often used by conventional starch extraction methods.

The economic usefulness of enzymatic hydrolysis as a method to recover starch from fibrous cassava residues relies on multiple practical factors, including cost, operational viability and the potential for scale-up. The enzymes cellulase and pectinase have high price points, particularly when used at industrial levels. The decline of enzyme production costs occurs because investigators continue to develop microbial fermentation techniques and enzyme stabilisation methods. The optimised combination of enzymes coupled with properly dosed enzymes according to this study decreases both material and processing expenses. The higher initial expenditures for enzyme-based extraction enable manufacturers to obtain purer starch extracts with no contaminants, which enhances their market value specifically for food and pharmaceutical industries27.

The system enables expansion while requiring small modifications to existing cassava processing facilities using tank reactors or bioreactors. Enzymatic processing operates at 40–50 °C under pH conditions of 4.5–5.5 to perform hydrolysis, which reduces operational requirements beyond thermal and chemical methods41. Enzymatic processing transforms industrial damaging materials such as peels or press cake into valuable co-products while achieving zero-waste management. The environmentally friendly approach optimises resource consumption and follows circular economy principles, which makes it appealing to companies looking for ways to minimise their carbon impact. The enzymatic process delivers parameter control accuracy which produces higher quality, standardised products than standard production techniques.

From an operational perspective, enzymatic processes are scalable, environmentally friendly, and more adaptable to automated or batch-controlled systems, making them attractive for industrial biorefineries and circular economy models42. In contrast, mechanical methods, while cost-effective and fast, often result in lower starch recovery rates because a significant portion of the starch remains embedded within fibrous tissues43. Furthermore, enzymatic processes have the potential to produce higher-quality products with fewer impurities compared to mechanical methods. This can lead to increased value and marketability for the final products in various industries.

Pasting characteristics of starch extracts from the cassava peel samples

The pasting properties of starches obtained from cassava peel samples treated with a combination of cellulase and pectinase enzymes are as shown in Table 3. Peak viscosity was lowest (98.17 RVU) for starch extracted from the cassava peel collected at AFC, which could indicate that this starch has a lower swell capacity as well as being more resistant to gelatinisation. On the other hand, the starch extracted from cassava peels from LMC and IIO had the highest peak viscosities (231.42 and 232.42 RVU, respectively). This means that it could absorb and swell more water, which is a normal trait of starches that are good at thickening. The OIR sample, with a peak viscosity of 160.08 RVU, demonstrated an intermediate swelling capacity compared to the other samples. The control sample displayed a high peak viscosity (225.25 RVU), suggesting that the extracted starches from LMC and IIO, in particular, perform similarly or even better than the control in terms of peak viscosity.

Table 3 Pasting characteristics of starch extracts from cassava Peel samples.
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Regarding trough viscosity, the starch extracted from the AFC cassava peel had the lowest value (94.83 RVU), indicating a low viscosity during the holding period, which could reflect limited shear-thinning behaviour. The LMC starch showed the highest trough viscosity (193.83 RVU), suggesting good stability under heat and shear conditions. The starches from OIR and IIO exhibited moderate trough viscosities (139.00 and 191.17 RVU, respectively), indicating relatively stable viscosities during prolonged heating. The control starch had a trough viscosity of 102.67 RVU, which is lower than that of LMC and IIO, indicating that these samples may have better heat stability than the control.

The starch extracted from cassava peel collected from AFC had the lowest breakdown viscosity (3.33 RVU), indicating strong paste stability under shear and heat, which can be advantageous in applications requiring high stability. In contrast, the starch extracted from LMC and IIO cassava peels exhibited higher breakdown values (37.58 and 41.25 RVU, respectively), suggesting a higher tendency to shear-thin under mechanical stress. However, a higher breakdown viscosity can also be indicative of good swelling properties. The OIR starch demonstrated an intermediate breakdown viscosity (21.08 RVU), indicating moderate stability, while the control had the highest breakdown viscosity (122.58 RVU), suggesting that the control starch is less stable than the enzyme-treated starches. Regarding final viscosity, the starch extracted from AFC cassava peel had the lowest value (127.67 RVU), consistent with its low peak and trough viscosities, indicating a less stable and cohesive paste upon cooling. In contrast, the starch extracted from LMC cassava peel had the highest final viscosity (283.00 RVU), suggesting it forms a very thick and cohesive paste after cooling. The starch from the OIR and IIO samples also had high final viscosities (193.25 and 255.58 RVU, respectively), indicating strong paste formation. The control sample showed a final viscosity of 151.67 RVU, which is lower than that of LMC and IIO, indicating less cohesive paste formation.

The starch extracted from AFC cassava peel had the lowest setback viscosity (32.83 RVU), indicating that it retrograded least. This could be considered beneficial in freeze-thaw stable products that require low starch retrogradation. The setback viscosity of LMC starch was the highest (89.17 RVU), reflecting considerable retrogradation, suitable for products making use of a tougher gel upon cooling. The setback viscosities of the OIR and IIO starches (54.25 and 64.42 RVU, respectively) were found to be moderate, suggesting intermediate interrogation. The control sample had a setback viscosity of 49.00 RVU, meaning it was closer in terms of retrogradation to OIR than LMC. The higher setback viscosities observed in LMC starch also indicated a greater tendency to retrograde, as was found by Yan, et al.44 and Marta, et al.45that starches with a higher content of amylose (or an alteration in the granular structure after enzymic treatment) tend to further retrograde upon cooling and form firmer gels.

The peak viscosity of 6.60 min observed for the starch extracted from cassava peel collected by AFC suggests a slower gelatinisation rate compared to others. On the other hand, starches in LMC, OIR, and IIO showed shorter peak times (5.60, 5.93, and 5.73 min, respectively), suggesting rapid gelatinisation, which may be useful for applications needing quick-cooking functionalities45. The control, with the shortest peak time of 3.80 min, indicates rapid gelatinisation. The AFC, LMC, OIR, and IIO starches have pasting temperatures all around 80 °C, the temperature at which each of these starts to gelatinise. The reference sample displayed a lower paste temperature (73.55 °C); it implies that the process of gelatinisation of this sample has begun at a low temperature compared to the starch obtained from the enzyme extraction method. Pasting temperature is a critical property that indicates the minimum cooking temperature required, influences production costs, and affects the stability of other ingredients during processing46. Higher pasting temperatures and longer holding times for enzyme-treated samples suggest a requirement for increased gelatisation temperature and extended cooking periods. However, choosing starches with shorter pasting times and temperatures is recommended for both technical efficiency and cost-effectiveness, as highlighted by Iwuoha47 and Baah, et al.48due to their quicker processing and lower energy consumption. Expanding on this, Tester and Karkalas49 demonstrated that starches treated with enzymes can alter their pasting profiles by modifying the amylose to amylopectin ratio and granular structure, leading to specific changes. Specifically, starches such as LMC and IIO, characterised by higher peak viscosities and lower breakdown viscosities, produce pastes with enhanced stability. These modifications result in improved functionality and versatility of the starches, making them suitable for a wider range of applications in various industries.

The peak viscosity also tells us how good the starch is at binding water, while the low stability of a starch paste is indicated by high breakdown values. Final viscosity characterises a specific starch by showing its stability in a hot paste during practical use and its ability to form various pastes or gels upon cooling50. In a study by Mohamed, et al.51it was found that modified starches exhibit varying pasting properties, with many showing increased peak and final viscosities as a result of enzymatic modification. This is because enzymes enhance the water absorption capacity of starches, leading to an increase in volume. This is also consistent with the higher final viscosities observed in LMC and IIO samples. In general, the results of the study showed that the recovered starch has useful properties, such as being able to hold more water and make pastes that stick better. These properties suggest that it could be used as a thickener in the food industry, as well as in the production of biodegradable films, medicinal binders, and paper and textile additives. The comparatively low gelatination temperature is advantageous for energy-efficient cooking and quick food compositions. The starch’s excellent film-forming capability and moisture retention make it a suitable material for biodegradable packaging films. This corresponds with the increasing demand for sustainable substitutes for petroleum-derived polymers. According to Das, et al.52 and Kapoor, et al.53enzymatically recovered starch is pure and functionally consistent, so it can be used to make tablets, fillers, or bio-based excipients in pharmaceutical formulations. In cosmetics, it functions as an absorbent or thickening ingredient. The sticky and viscoelastic characteristics can be utilised in paper coating and textile sizing, where starch enhances surface smoothness and strength. The value-added applications enhance the efficacy of enzymatic recovery from cassava waste as a pathway to industrial sustainability.

Significant variations on pasting qualities of the hydrated starch extracted from cassava peels after treatment with enzymes were observed as a function of sample and enzymatic treatments. Enzymatic modification resulted in higher peak viscosity and a more stable viscosity during holding and cooling phases. The LMC and IIO samples that had higher peak, final, and setback viscosities in the pasting profiles showed that they could be used in situations where the paste needs to be thickened or stable. The control sample showed rapid gelatinisation with decreased stability, whereas the AFC sample displayed the least stable paste with the lowest viscosities. These results are in line with earlier research on starches treated with enzymes, which usually shows improved capacity for swelling and water absorption but variable degrees of paste stability and retrogradation. Lower setback viscosity obtained in the extracted starch from AFC compared with the control implies reduced retrogradation. This is advantageous for frozen foods, sauces, or bakery items requiring prolonged shelf life and textural retention.

In general, the results showed that the enzyme-modified starch was better than the native cassava peel starch at improving viscosity and thermal stability and reducing degradation, all of which are important for high-performance industrial uses. These enhancements are likely due to partial hydrolysis of complex polysaccharides and reduced structural constraints, which enhance gelation and paste clarity. This positions enzyme-treated cassava peel starch as a more functional and versatile ingredient in food, pharmaceutical, and biodegradable packaging industries.

Effect of methanol on citric acid production by A. niger on cassava peels

The effect of methanol on citric acid production by A. niger using cassava peel after 72 h of growth at 30 °C is illustrated in Fig. 4. Initially, citric acid yields are relatively low across all trials, with values of 0.79 g/L (AFC), 0.75 g/L (LMC), 0.72 g/L (OIR), and 0.70 g/L (IIO). These baseline yields represent the natural citric acid production capacity of A. niger without the enhancement provided by methanol. A noticeable increase in citric acid yield is observed with the addition of 1% methanol, resulting in yields of 0.83 g/L (AFC), 0.95 g/L (LMC), 0.79 g/L (OIR), and 1.05 g/L (IIO). This suggests that even at a low concentration, methanol has a positive impact on citric acid production. Further increases in methanol concentration to 2% result in higher citric acid yields: 1.04 g/L (AFC), 1.15 g/L (LMC), 0.86 g/L (OIR), and 1.23 g/L (IIO). This indicates that methanol continues to stimulate citric acid production, likely by enhancing metabolic activity or altering the metabolic pathways to favour citric acid synthesis.

There are several theories to explain the stimulating effect of methanol on citric acid production. Hang and Woodams54 said that the rise in citric acid production might be because mycelial growth slowed down, which made it easier for glucose to be used for making citric acid instead of growing cells. Moreover, organic solvents such as methanol increased the permeability of cell membranes, and thus citric acid was secreted across membranes55,56. Hence, organic solvents can improve citric acid formation via microbial fermentations.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Effect of methanol on production of citric acid from cassava peel by A. niger.

The optimal citric acid yields occur with 3% and 4% methanol, reaching 1.41 g/L (IIO) at 3% and 1.42 g/L (IIO) at 4%. However, in some instances, the yield plateaus or shows only slight increases between 3% and 4% methanol (e.g., IIO: 1.41 g/L vs. 1.42 g/L). This indicates that while higher methanol concentrations can enhance citric acid production, there might be a threshold where additional methanol does not significantly boost the yield further. Navaratnam, Arasaratnam and Balasubramaniam55 said that methanol might raise the production of citric acid at low concentrations by speeding up the metabolic process in a way that helps make citric acid and slowing down the production of unwanted byproducts. Researchers, Haq, Ali, Qadeer and Iqbal56 and Ajala, Adeoye, Olaniyan and Fasonyin19 also found that adding methanol to the fermentation medium greatly increased the production of citric acid, with the best results happening at a concentration of about 3% (v/v). Exceeding this concentration, the yield enhancement often stabilises. The stabilisation of citric acid yield beyond this concentration arises as metabolic redirection, membrane permeability, and enzyme activity attain respective thresholds, inhibiting further improvement.

Furthermore, the plateau in citric acid production seen at 3–4% methanol likely shows a limit beyond which disrupting membranes and stopping enzymes from working outweigh the benefits of higher permeability. Low amounts of methanol enhance citric acid excretion by augmenting membrane fluidity; however, elevated concentrations may jeopardise membrane integrity and enzyme activity, hence restricting additional yield improvements. These findings highlight the necessity of optimising methanol dosage to achieve a balance between increased metabolite release and cellular survival. Thus, based on the results of this study, a methanol concentration of 3–4% is deemed as the most effective for citric acid yield maximisation. The concentration, however, is specific and will depend on a variety of process conditions, such as pH, temperature, and strain for A. niger. Exploration of possible synergies of methanol with other potential stimulants or fermentation conditions could give rise to increased yields or more sustainability. Additionally, due to the potential high toxicity of methanol and its complicating effect on alcoholic fermentation, it is important to study the long-term effects of methanol as a stimulant in citric acid production for sustainability and product-end use perspectives.

The usage of methanol successfully increases citric acid excretion but brings multiple essential safety hazards. Nagendran, et al.57 affirmed that exposure to low doses of methanol through inhalation, ingestion or skin absorption causes fatal neurological harm to humans due to its high toxicity levels. The industrial applications of methanol need thorough safety measures that combine ventilation systems with personal protective equipment and fire protection measures because it is both flammable and volatile. Methanol procurement, along with storage procedures and regulatory adherence, can become economic barriers when operating at larger production scales, mainly affecting low-resource areas58. Therefore, given these concerns, optimising methanol concentration and exploring safer alternatives or controlled dosing strategies is essential for industrial scale-up. This will ensure that the benefits of methanol-based processes can be realised without compromising safety or sustainability.

Effect of temperature on citric acid production by A. niger on cassava peels

The incubation temperature is essential for citric acid production by A. niger due to its effect on all physiological activities of the microbes59. The results of the effect of temperature on citric acid production by incubating the fungus at different temperatures (25 °C, 30 °C, and 37 °C) for a period of seventy-two hours with 3% methanol are given in Fig. 5. The production of citric acid at 25 °C is lower in all the experimental studies, including 0.42 g/L in AFC, 0.48 g/L in LMC, 0.38 g/L in OIR, and 0.43 g/L in the IIO. Incubation at 25 °C is evidently not suitable for producing citric acid by A. niger compared to 30 °C. The citric acid yield for 30 °C was 1.43, 1.54, 1.36, and 1.49 in LMC, AFC, LMC, OIR, and IIO, respectively. In contrast, citric acid yields were much higher at 30 °C (optimal temperature), with maximum values of 1.43 g/L for AFC, 1.54 g/L for LMC, OIR for 1.36 g/L, and IIO for 1.49 g/L. This implies that 30oC conditions are optimum for citric acid production under these experimental parameters. This temperature can improve the biosynthesis of citric acid by increasing metabolic reactions and enzyme activities involved in the synthesis. The concentration of citric acid produced is reduced at 37 °C, with values decreasing to 0.84 g/L, 0.98 g/L, 0.79 g/L, and 0.89 g/L, respectively, for AFC, LMC, OIR, and IIO. This temperature could indeed be too high, hence causing thermal stress or denaturation of essential enzymes involved in the citric acid production. Thus, based on this study, the optimum incubation temperature for citric acid production in A. niger should be around 30 °C.

Haq, Ali, Qadeer and Iqbal56 also noted that the A. niger enzyme system is denatured at higher temperatures. Nampoothiri, et al.60 reported that slow metabolism, enzyme denaturation, and low viability could also inhibit the production of citric acid. Several authors reported an optimal incubation temperature between 28 and 32 °C for the production of citric acid; for instance, Ali, et al.61 and Asad-Ur-Rehman., et al.62 reported the optimal production at 30 °C, attributing this to optimum functioning of enzymes and cellular metabolism. In contrast, Roukas63 and Haq, Ali, Qadeer and Iqbal56 found that further increases in the incubation temperature, up to 37.5 °C, were detrimental with respect to the production of citric acid competing with butyric and propionic acids, possibly due to acetic and lactic repressed culture growing. Dashko, et al.64 also noted that temperatures very much above the optimum will result in rapid fermentation together with excessive growth oxidation of sugar to CO2 at high rates. As a result, citric acid concentrations decrease, while oxalic and gluconic acids increase, in addition to the denaturation of citrate synthase.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Effect of temperature on production of citric acid from cassava peel by A. niger.

Furthermore, Abu Bakar, et al.65 reported that at elevated temperatures, fungal metabolism is negatively affected through a combination of enzyme denaturation, impaired membrane transport, and activation of stress responses that divert energy from citric acid biosynthesis. These conditions can destabilise key metabolic enzymes and reduce biomass growth, ultimately inhibiting citric acid yield. Maintaining an optimal temperature range is therefore critical to preserving metabolic efficiency and process performance. In addition, it is important to carefully monitor and control temperature fluctuations during the fermentation process to ensure consistent and high citric acid production. Failure to do so could result in decreased productivity and overall efficiency of the bioprocess.

The results of this work highlight the importance of keeping an incubation temperature close to 30 °C during industrial fermentation for obtaining the highest citric yields. Although the results presented here show that temperatures of 30 °C could be optimal in these conditions, future studies will test for interactions with pH levels and nutrient concentrations or methanol supplementation to further improve this process. Similarly, it may also be interesting to investigate how different temperatures influence the activity of enzymes that participate in citric acid synthesis, which can contribute to a better understanding of fermentation. Understanding these dynamics may help in developing more efficient and cost-effective approaches for citric acid production on an industrial scale.

Conclusions

This study highlights an integrated bioprocessing approach for the valorisation of cassava residues, demonstrating both environmental and economic benefits. The key outcomes from the study were presented:

  • Charcoal filtration was the most effective method for reducing BOD₅ and HCN in cassava effluents.

  • Despite improvements, BOD₅ and HCN levels were not yet within safe discharge limits; thus, further treatment is needed to meet international discharge standards (e.g., WHO, EPA).

  • Enzyme type and extraction time significantly affected starch yield from cassava peels. A cellulase–pectinase combination achieved the highest starch recovery, showing synergistic enzyme action. While longer extraction times consistently led to higher starch yields.

  • Starches from LMC and IIO samples had superior pasting properties, particularly higher peak, final, and setback viscosities, making them suitable for stable thickening applications in the food or bio-based materials sectors. Conversely, AFC starch was less stable, and the control sample showed rapid gelatinisation but limited paste stability.

  • The fermentation studies demonstrated that methanol supplementation (1–4%) significantly enhanced citric acid yields, validating cassava peel as a low-cost feedstock for biotechnological production. However, yields plateaued beyond 3% methanol, likely due to membrane or enzyme inhibition, highlighting the importance of dosage optimisation.

  • Further research is needed to optimise fermentation conditions and explore cost-effective scale-up strategies. Also, integrating charcoal filtration with constructed wetlands, biofilters, or anaerobic digestion to achieve full compliance with BOD₅ and HCN discharge limits could be evaluated.

  • Detailed mechanisms of methanol’s action on fungal systems and feasibility of scale-up, including cost, safety, and regulatory aspects, can also be evaluated. These suggested advances will further enhance the economic viability and sustainability of integrated biorefineries using cassava residues.