Nepenthes mirabilis Pitcher Fluid Functionality for Agro-Waste Pre-Treatment: Effect of pH, Temperature, Trace Element Solution and the Pore Size of the Waste

Abstract

Nepenthes mirabilis pitcher fluid is known to hydrolyse lignocellulosic mixed agro-waste (MAW) into fermentable sugars through a cocktail of oxidative and hydrolytic enzymes. However, the influence of factors such as pH, pore size, temperature and trace elements on its functionality is not adequately understood. This study aims to explore the potential of Nepenthes mirabilis pitcher fluid for the pre-treatment of MAW (>106 µm) by assessing the influence of the factors mentioned above on the yield of total reducible sugars (TRSs). The association between the trace element solution, pH, and temperature was evaluated using standard methods: Dinitrosalicylic acid (DNS) assay for the concentration of TRSs, and BET assay for the surface area and pore properties of the samples. The results showed that the highest concentration of TRSs (407.50 g/L) was at pH 2, albeit below ambient temperature, while pores (>106 µm) of agro-waste can accommodate <10 kDa enzymes, i.e., the enzymes could be adequately embedded within the pores of the milled agro-waste used. In conclusion, supplementing the pitcher fluids with a trace element solution did not improve the yield of TRS, but a low pH at below ambient temperature was more effective.

1. Introduction

Nepenthes spp. has “monkey cups” filled with an acidic enzyme containing fluid, hereafter referred to as pitcher fluid, capable of degrading leaf litter, including insects, into useable nutrients that serve as a source of energy for the plants’ wellbeing and reproduction. Pitcher fluids produced by Nepenthes spp. have distinctive characteristics suited for agro-waste pre-treatment. Since the pitcher fluid is highly acidic, i.e., with a pH range of 1.8–2 [1], it can be responsible for the delignification and decoupling of cellulose and hemicellulose chains into fermentable sugars. A handful of researchers have reported on the constituents of the fluids from a variety of Nepenthes spp.; however, there is still little evidence to suggest that such acidic fluid can be applied for biomass pre-treatment at a large scale in biorefineries [2,3,4]. Additionally, current investigations have proven that the pitcher fluid does have lignin–holocellulose hydrolysing enzymes [5]. However, their functionality outside the “monkey cups” can be enhanced by varying environmental conditions to suit large-scale usage and efficacy. These environmental conditions or factors include but are not limited to pH, temperature, and supplementation with a cofactor solution hereafter referred to as a the trace element solution.

Pre-treatment of lignocellulose waste is generally influenced by pH, temperature, and holding times [6]. Generally, hydrolysate quality is directly impacted if the conditions are unsuitable. Overall, preparation of fermentable hydrolysates, pre-treatment of waste feedstock, pH, temperature, and to some extent, trace elements in varying proportions can produce hydrolysates that enhance a fermenter’s performance and, thus, its efficacy. Some researchers suggest that when you have lignocellulosic waste, a pH of 2 and a temperature of 33 °C provide adequate conditions for pre-treatment [2,5,7]. Nepenthes mirabilis pitcher fluid with a pH range of 2 to 2.9 under ambient temperature conditions (from 15 to 25 °C or up to 30 °C) can satisfy these requirements. When the pre-treatment of agro-waste is performed using pitcher fluids containing a cocktail of enzymes, such as those from Nepenthes spp., it can be advantageous for processes that focus on eco-friendliness without using chemicals. It was further suggested that pre-treated agro-waste at lower pH improves the solubilisation of hemicellulose [7,8,9].

Since it was determined that N. mirabilis pitcher fluids contain enzymes, such as β-1,3-Glucanase, putative peroxidase 27, and Thaumatin-like protein, among others [7], their functionality must be determined by focusing on pH, temperature, and co-factors in the form of trace elements. Some enzymes were determined to be stimulated by Ca²⁺, Mg²⁺, and Mn²⁺ [10,11]. There is a suggestion that metal salts, including compounds such as EDTA, can improve the activity of some enzymes, such as endoglucanase, exoglucanase, and β-glucosidase, which are some of the important enzymes in agro-waste pre-treatment [12]. Those associated with N. mirabilis, such as purple acid phosphate, have improved functionality under acidic pH while having no specific co-factor preference. However, supplementing a minute concentration of Mg²⁺, Ca²⁺, and Mn²⁺ to Class IV chitinase increases the enzyme’s activity [12]. That is what motivated the exploration of using a trace element solution. Therefore, the interlinkages between pH, temperature, and a trace element solution during milled agro-waste pre-treatment, as used in this study, must be assessed using N. mirabilis pitcher fluid.

Another aspect that needs to be assessed is the relation between the porosity of the MAW and the enzyme fraction (<10 kDa) determined to be suitable for the pre-treatment of the milled mixed agro-waste used in this study [7]. This is because if the pore size of the milled agro-waste is large enough in comparison to the size of the enzymes, the enzymes in the pitcher fluid can adequately be embedded in the pores of the agro-waste. Therefore, improving the yield of targeted products, mainly total reducible sugars, improves the pre-treatment process. This linkage has not been studied before.

Therefore, the aim of this study was to (1) assess the influence of pH, temperature, and trace element solution on the pre-treatment of the milled agro-waste using N. mirabilis pitcher fluids and (2) to assess the link between the porosity of the used agro-waste to the fraction (<10 kDa) of the pitcher fluid enzymes. Previously, it was determined that a 72 h holding time, a particle size of >106 µm, and an enzyme fraction of <10 kDa resulted in the highest production of total reducible sugars, while total phenolic compounds were low using N mirabilis as a sole pre-treatment agent [7]. Therefore, these conditions were adopted for this study, specifically assessing TRSs in hydrolysates produced from the pre-treatment of agro-waste.

2. Materials and Methods

2.1. Collection and Processing of the Mixed Agro-Waste

Mixed agro-waste with components such as Malus domestica (apple) peels, Quercus robur (oak) yard waste, Citrus sinensis (orange) peels, Vitis vinifera (grape) pomace, and cobs from Zea mays (maise) was collected from a fruit and vegetable store in Zonnebloem, Cape Town, South Africa, with Quercus robur (oak) yard waste (leaves) being collected from the Cape Peninsula University of Technology (CPUT), District Six Campus (Western Cape, Cape Town, SA, USA). After mixing with a pre-rinsing step, the agro-wastes were dried at 80 °C for 24 h. This was extended to 72 h due to the adhesive nature of the C. sinensis peels. Following this, screening to obtain a > 106 µm particle size was carried out. Mixing the agro-waste demonstrated that it is cost-effective and thus produces more quality hydrolysates than single feedstocks [13]. As in previous experiments [2,7], 20% (w/w), i.e., 10 g per agro-waste, was used.

2.2. Collection and Sample Fractionation of the N. mirabilis Pitcher Fluid

The N. mirabilis pitcher fluids were collected from plants cultivated at Pan’s Carnivores Plant Nursery (21 Kirstenhof, Tokai, Cape Town, South Africa) and stored in ice. To remove debris, a centrifugation regime was applied whereby conditions were set at 4000× g for 15 min with subsequent filtration at 0.22 µm using Millipore membrane filters (Merck, Burlington, MA, USA). To obtain the <10 kDa, a filter with size <10 kDa was used, with the filtrate being used for the experiments. This process was conducted using a centrifuge at 4000× g for 10 min. After that, the filtrate was stored at −20 °C, which was determined to be an appropriate storage temperature before use in the experiments. The filtrate was used as it is after thawing.

2.3. Trace Element Solution Preparation for Pre-Treatment Supplementation

The trace element solution used in this study was prepared using analytical grade chemicals, i.e., by dissolving 1.5 g of Nitrilotriacetate in 800 mL sterile distilled water with the pH being adjusted to 6.5 using 1 M KOH (8 g/500 mL). Thereafter, the following com-pounds were added to the Nitrilotriacetate: ZnSO₄·7H₂O (0.1 g), FeSO₄·7H₂O (0.1 g), MgSO₄ (3 g), MnSO₄ (0.5 g), NaCl (1 g), CuSO₄ (0.1 g), AlK(SO₂)₂·12H₂O (0.01 g), H₃BO₃ (0.01 g), Na₂MnO₄·2H₂O (0.01 g), MgSO₄·7H₂O (6.14 g), MnSO₄·H₂O (0.56 g), CoCl₂·6H₂O (0.187 g) and CoCl₂ (0.1 g), with the solution being made up to 1000 mL [14]. The solution was filter-sterilised using a 0.22 µm filter and autoclaved. It was then stored at 4 °C before use, as previously elucidated.

2.4. Conditions for Pitcher Fluid Facilitated Mixed Agro-Waste Pre-Treatment

The agro-waste was slurried at 5% (w/v) in 100 mL Schott bottles with 10 mL of the <10 kDa pitcher fluid of the slurry being used. Subsequently, the pH was adjusted using either HCl or KOH to attain the required pH of the slurry. The variation in the volume of the trace element solution to be used is shown in

Table 1. Experimental runs used in the study.

Runs	pH	Temperature (°C)	Trace Element Solution (µL)
1	12	25	50
2	10	16	-
3	2	16	-
4	2	16	100
5	6	25	50
6	2	33	-
7	10	16	100

. A shaking (120 rpm) incubator (LABWIT ZWY-240, Shanghai Zhicheng Analytical, Shanghai, China) was used to vary the temperature. Sampling (3 mL) was carried out only at 72 h as this was the incubation time previously determined [7] to be suitable to produce the highest total reducible sugars. All samples collected were centrifuged at 4000× g for 10 min, and the debris-free supernatant was used to quantify the total reducible sugars. Table 1 indicates the experimental conditions used per run (n = 7). The values for the individual runs (n = 7) were analogous to those generated by the artificial neural network using MATLAB (Version 2017B), as shown in Table 1. All the runs were performed in triplicate.

2.5. Quantification of Total Reducible Sugars from Agro-Waste Pretreatment Hydrolysates

The dinitrosalicylic acid (DNS) assay protocol of Miller [15] was used to determine the concentration of total reducible sugars. The method’s basis is that DNS is reduced to 3-amino-5-nitrosalicylic acid, although different sugars yield different colours. This assay tests for the presence of the free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present to the corresponding acid while DNS is simultaneously reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions. The formation of 3-amino-5-nitrosalicylic acid results in a change in the amount of light absorbed at wavelength 540 nm. The absorbance measured using a spectrophotometer is directly proportional to the amount of reducing sugar. Since the measurements performed in this study are for total reducible sugars in a mixture, the colourimetric measurements were expected to be consistent. A Jenway 7305 UV/Vis spectrophotometer (Cole–160 Parmer, Staffordshire, UK) was used for this assay. A calibration curve of absorbance value against known concentration (glucose standard) was needed to calculate the concentration in the samples. The calibration curve correlation coefficient (R²) was 0.95. All measurements were performed in triplicate, and the averages were used in data analysis.

2.6. Determination of the Milled Agro-Waste Porosity Using the Brunauer–Emmett–Teller (BET) Method

The surface area and pore properties of the samples were evaluated using a Mi-cromeritics 3Flex adsorption analyser (supplied by Poretech, Roodepoort, South Africa), with N₂ as an adsorbate, with a bath temperature of −196.9 ℃ using sample mass of 1.67 g of the agro-waste with equilibrium intervals of 15 s. The analysis-free and ambient-free spaces were 48 and 15 cm³, respectively. Before sample analysis, all the samples were dried in a vacuum oven at 105 ℃ for 3 h before a representative of the samples was loaded into the sample tubes. The degassing of the samples was then conducted following the method described elsewhere. Briefly, the samples were degassed at 300 ℃ for 12 h and further at 45 ℃ for another 12 h before switching to low-pressure gas adsorption (LPGA) analysis, using N₂ and CO₂ as adsorptive gases. The LPGA was conducted at –196 ℃ in liquid N₂ for N₂ adsorption and 0 ℃ in an ice-water bath for CO₂ adsorption. Adsorption isotherm data were captured automatically from the equipment and analysed on the Mi-crometrics Microactive v5.02 platforms. Details of the data processing and surface areas and pore properties evaluations are described in Okolo et al.’s work [16].

3. Results and Discussion

3.1. Pre-Treatment of the Agro-Waste Using N. Mirabilis under Different pH, Temperature, and Trace Element Solution Conditions

Alkaline pre-treatment has been said to be the most efficient pre-treatment strategy as it rapidly solubilises lignin. However, this pre-treatment strategy might be suited to pre-treat agro-waste with a high lignin content. Dlangamandla et al. [2] have previously determined that the agro-waste used in the study has a lignin content of 27%, while that of holocellulose accounted for 73%. This distribution in constituents between lignin and holocellulose suggests that alkaline pre-treatment might be unsuitable. Additionally, the impression that corn cob has a higher lignin content because it is hardy is misleading. Orange and apple peels, including grape pomace, have a higher lignin content [17,18], which is greater by 5 to 6% compared to a corn cob. Similarly, the cellulose content of orange peel and grape pomace is higher than that of corn cob and apple peel. In this study, the observation was that a pH of 2 below ambient temperature produces a higher concentration of total reducible sugars than when the temperature is higher (>−25 °C), particularly at 33 °C. It was expected that a lower pH and a high temperature (33 °C) could improve reactivity between the N. mirabilis pitcher fluid and the agro-waste; however, this was not evident. A higher temperature might likely have deactivated the constituents in the pitcher fluid as these function without heating in the natural habitat of the N. mirabilis plants. Even though alkaline (with a high pH) pre-treatment is reportedly an efficient method, low total reducible yields were observed at pH levels ranging from 10 to 12 in this study.

Table 2. Production of total reducible sugars under varying pH, temperature, and trace element solution.

Runs	pH	Temperature (°C)	Trace Element Solution (µL)	Total Reducible Sugars (g/L)
1	12	25	50	65.00
2	10	16	-	231.25
3	2	16	-	407.5
4	2	16	100	407.02
5	6	25	50	148.75
6	2	33	-	141.88
7	10	16	100	202.50

further indicates that supplementing the pre-treatment regime with a trace element solution had minimal influence on the yield of total reducible sugars in the hydrolysates.

This might suggest that the N. mirabilis pitcher fluid might contain sufficient metallic ions; thus, supplementing the pitcher fluid with additional metallic ions might have some-how inhibited the functioning of the cocktail of enzymes contained therein. Therefore, further exploratory studies are needed to ascertain the metal ion content of the pitcher fluid. Judging from the literature, not many researchers have worked on N. mirabilis pitcher fluid for agro-waste pre-treatment. However, several researchers have reported on the pre-treatment of agro waste using other biological materials, mostly microorganisms such as fungi [19,20,21,22]. In ref. [20], a variety of microorganisms (mostly fungi) produced cellulolytic enzymes cocktail for a high yield of reducing sugars, which was blended and tested on various lignocellulosic agro-waste. Their results showed a maximum TRS yield of 56.43 mg/g after 48 h of enzyme pre-treatment. This result is much lower than the TRS yield of 407.50 g/L obtained in the present study, although the conditions are a bit different. The authors did not take into account various factors such as pH, temperature and trace elements when pre-treating mixed agro-waste as proven in the present study. Another related study [19], produced multi-lignocellulolytic enzymes isolated from the B. licheniformis strain 2D55 including xylanase and β-glucosidase suitable for degradation of complex lignocellulosic substrates. Their results showed a maximum TRS yield of 79.8 U/mL; again, these are much lower than the present study, which further supports our hypothesis. Our idea is that there is a need to take into account various factors such as pH, temperature and trace elements as well as pore sizes when pre-treating mixed agro-waste with biologically derived enzymes.

3.2. Correlation between the Milled Mixed Agro-Waste Porosity and Pitcher Fluid Efficacy Conditions

Researchers report that preparatory methods increase the porosity of the agro-waste [23], which then enhances enzyme accessibility to some extent. This is achieved using numerous techniques, some of which are physical, e.g., milling, as used in this study. In-creasing the porosity of the agro-waste, in turn, increases the specific surface area. If not appropriately managed, agro-waste morphology, including roughness, can influence the waste’s hydrolysis and impede the production of total reducible sugars and usable hydrolysates. It is suggested that low enzyme loading or quantities can be utilised if a highly porous agro-waste is used as feedstock [24]. There is also a correlation between the size (kDa) and shape of the enzyme and the average pore size (nm) to which it can attach itself in the agro-waste. Such a correlation is hardly ever reported. It is estimated that the following enzyme sizes are associated with a minimum pore diameter, i.e., 5 kDa (2.2 nm), 10 kDa (2.84 nm), 20 kDa (3.56 nm), 50 kDa (4.8 nm), 100 kDa (6.1 nm), 200 kDa (7.68 nm), 500 kDa (10.42 nm) [25], with 10 angstroms (Å), a unit measurement from BET, being equivalent to 1 nm.

Since <10 kDa (associate size of 2.84 nm) pitcher fluid fraction was identified as being suitable to pre-treat the milled agro-waste (>106 µm) used in this study, its average pore size of 2.84 nm (28.4 Å) would thus also explain why the fraction performed satisfactorily, as it also contains a cocktail of enzymes associated with agro-waste decomposition [7].

Table 3. Adsorption and desorption pore diameter.

Parameter	Value (Å)	Value (nm)	Estimated kDa
Adsorption average pore diameter	79.90–76.91	7.99–7.68	200
Desorption average pore diameter	71.77–71.46	7.18–7.15	≤200

lists pore diameter values, while Figure 1A represents the isotherm plot from the BET measurements. Additionally, the BET surface area (see Figure 1B) for the >106 µm agro-waste was determined to be 0.6458 m²/g.

In most instances, the preparation of agro-waste when using milling, as in this study, increases its porosity and surface area [26]. It was also mentioned that different preparation techniques for the agro-waste can result in variations in its structure, which can then affect hydrolysis, with pore volume being majorly influential in hydrolysis [27], thus explaining the production of hydrolysates with a high content of total reducible sugars. In this study, the single-point adsorption total pore volume was 0.001242 cm³/g. Figure 1C illustrates the cumulative pore volume of the milled agro-waste used in this study. In general, milling the agro-waste will reduce the mixed waste, i.e., particle size, thus increasing the pore volume, as indicated elsewhere [28]. Barakat et al. [29] have criticised the use of methods such as BET, suggesting that they are not precise, especially when macropores are present in the samples. However, the BET method used herein was deemed sufficient. Although it is understood that milling the agro-waste before pre-treatment with N. mirabilis pitcher fluid can assist in the effectiveness of hydrolysis, others advocate for wet milling [30]; however, this generates another waste stream.

In summary, agro-waste pre-treatment is presently carried out in a number of processes, in which deligno-cellulolysis of the waste to fermentable sugars is facilitated, albeit producing inhibitors associated with the souring of downstream fermentations, including enzymatic hydrolysis. The development of alternative and environmental benign holocellulose valorisation methods for the pre-treatment of agro-waste, for the production of value-added products, while limiting the production of toxicants from the lignin component of the waste, is necessary. This will provide a new promising alternative strategy towards the sustainable and efficient processing of numerous agro-waste types [31,32]. In the present study, the Nepenthes mirabilis plant’s digestive fluid was proven to be effective in targeting holocellulose extraction under optimal conditions of low pH, ambient temperature, trace element solution and porosity of agro-waste. The plant digestive fluids contained digestive enzymes which have the potential to biodegrade complex and polymeric molecules [33]. This pre-treatment method requires less energy as it was operated at ambient temperature, and it eliminates the use of hazardous chemicals such as dilute inorganic acids; however, the digestive fluid is acidic, with an added advantage of reducing the production of inhibitory compounds such as phenolics [34]. When a high temperature is used for agro-waste pre-treatment, there is a risk of TRSs decomposition, which leads to the formation of Levoglucosan, a six-carbon-ring compound generated when carbohydrates are pyrolysed [35]. The acidic nature of the N. mirabilis pod juices, which also facilitate leaf litter digestion for the plant’s nutrition [36], can be used to pre-treat mixed agro-waste for TRS production from a variety of lignocellulose-containing biomass.

4. Conclusions

In this study, we further demonstrated that it was prudent to utilise N. mirabilis pitcher fluid with a pH range of 2–2.09 to pre-treat the milled mixed agro-waste at ambient temperature, as the highest concentration of total reducible sugars (407.50 g/L) was deter- mined to be at pH 2, albeit below ambient temperature. The <10 kDa enzyme fraction was suitable as the pore size, i.e., the pore diameter of the agro-waste. The observed BET sur- face area of 0.6458 m²/g was hypothesised to be suitable for effective enzyme-facilitated hydrolysis. The role of the trace element solution was non-evident, with a recommendation that metallic species in the pitcher fluid be investigated in future studies.