Role of Biotransformation of Acacia nilotica Metabolites by Aspergillus subolivaceus in Boosting Lupinus termis Yield: A Promising Approach to Sustainable Agriculture

: Biotransformation plays a signiﬁcant role in sustainable agriculture. This process involves utilizing microorganisms, such as bacteria and fungi, to transform organic compounds and metabolites into bioactive compounds which have beneﬁcial effects on plant growth, yield, and soil characters. Accordingly, the present study aims to explore the role of biotransformation of Acacia nilotica metabolites by Aspergillus subolivaceus in boosting L. termis yield, as an important strategy in agricultural sustainability. A pilot experiment was performed on ﬁve fungal strains ( Fusarium oxysporium A. aculeatus , Aspergillus. subolivaceus , Rhizopus oryzae and Trichoderma viride ) which were grown on different parts of plants ( A. nilotica leaves; green tea leaves, green pepper fruits and pomegranate fruits), and the results indicated that the most active metabolite for the growth of L. termis seeds was the fungal metabolite of A. subolivaceus growing on A. nilotica . More speciﬁcally, we assess how metabolites produced by Aspergillus subolivaceus using A. nilotica leaves affect the biochemical properties and chemical composition of L. termis seeds. A. subolivaceus was grown on leaves from A. nilotica to obtain metabolites and fractionated into four extracts. Two concentrations of each extract were examined by pretreating the seeds of L. termis . The study found that all four extracts contributed to an increase in yield and some biochemical properties of the yielded seeds. The best results were obtained by treating the L. termis seeds with an extract obtained from diethyl ether, which led to a signiﬁcant increase in total nitrogen, amino nitrogen, glucose and protein contents of the seeds. According to 1 H NMR guided GC/MS analysis, our results showed an increase in phytochemicals such as terpenes, fatty materials, and ﬂavonoids including 3 (cid:48) ,4 (cid:48) ,7-trimethoxyquercetin and 4-methyl-p-menth-8-en-3-one, which have not been stated before from A. nilotica suggesting that biotransformation may have occurred due to the presence of A. subolivaceus .


Introduction
The significance of biotransformation in sustainable agriculture cannot be ignored or disputed. This process, which includes the use of microorganisms to convert organic compounds into beneficial substances for plant growth and soil characteristics, has a Table 1. GC/MS study of Acacia nilotica and a review of related literature.

Microorganisms
The fungal strain was subordinated in order to perform complete identification using one of the most advanced sophisticated installations; an imaging analysis system using soft-imaging software (AnalysisPro ver.3.0) at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Egypt. Solid state fermentation medium (SSF): SSF medium was prepared [24]. Fungal strains were grown on PDA slants for 7 days at a temperature of 30 • C, the spore suspension was prepared by adding 10 mL of sterile basal medium. 50 mL of the sterile substrate in a 250 mL conical flask was inoculated by 2 mL (2 × 10 7 spores) spore suspension.
The fungal strains (A. aculeatus, Trichoderma viride, A. subolivaceus, Rhizopus oryzae and Fusarium oxysporium) were grown on different plant parts (A. nilotica leaves; green tea leaves, pomegranate fruits and green pepper fruits). The fungal metabolites attained were tested by treating L. termis seeds. Fungal metabolites gained from the growth of A. subolivaceus on A. nilotica showed the finest results. So, this fungal metabolite was fractionated by diethyl ether and ethyl acetate into four fragments which were used for treating the Lupinus seeds.

Preparation of Metabolites
The purity of metabolic extract was confirmed by centrifugation at 15,000 rpm for 20 min, to ensure full removal of fungal culture.
(a) Water extract: Was carried out according to [25]. (b) Total diethyl ether extract was obtained according to [25,26]. Additionally, the aqueous extract is obtained by resuspension, shaking, and filtration of the residual substrate after the diethyl ether extract in distilled water. (c) Total diethyl ether extract separation according to [27]. Using a separation funnel, the diethyl ether extract was separated into two layers, the aqueous layer was re-extracted by ethyl acetate. However, the final diethyl ether extracts and ethyl acetate were used for Lupinus seeds treatments.

Experimental Design
L. termis seeds were surface sterilized by being submerged in a 0.01% HgCl 2 solution for three minutes. After that, seeds were split into 100-seed bunches that were each equally sized. Each group was pre-soaked for 10 h in two different concentrations (50-100%) of one of the obtained fungal metabolite extracts (water, diethyl ether, ethyl acetate, and aqueous extract) before seeding. Additionally, one group served as control by soaking for the same amount of time in regular water. All seedlings were grown in pots measuring 30 cm in diameter, which contained uniformly distributed soil in a ratio of sand to clay of 1:2 (vol/vol). The plants were exposed to 11 h of light and 13 h of darkness (normal day-night conditions) and temperatures was 32 • C ± 2 and 20 • C ± 2 for day and night, respectively. The relative humidity was 58% during the growth period. The yield was recorded on the harvest day at 106 days from sowing and the analysis was done on the dried seeds that had been handled differently and not at all.

Statistical Analysis
The data were subjected to analysis utilizing L.S.D. test at the probability threshold of 0.05. ANOVA analysis was performed using IBM statistics software version 20 [28].

Estimation of Carbohydrates
The extraction techniques used for the various carbohydrate fractions put to the test were primarily those of Yemm and Willis [31] and Handel [32]. The o-toluidine method of Fetris [33], which was modified by Riazi et al. [30], was used to assess the glucose contents. A modified version of Handel's procedures was used to determine the amount of sucrose in the sample [32]. The amount of total soluble sugar was calculated using a modified version of Yemm and Willis' procedures [34]. Polysaccharides was estimated by the methods of Sadasivam and Manickam method [35].

Estimation of Nitrogenous Constituents
The Yemm and Willis method [36] was used as a basis to estimate the nitrogenous constituents, with some modifications. To estimate ammonia-N, the method described by Delory [37] and modified by Naguib [38] was utilized, with spectrophotometry and Nessler's reagent employed. Naguib [38] suggested the technique used to estimate amide-N. By Muting and Kaiser [39], a method for estimating amino-N was developed. The traditional semi-micropropagation of the Kjeldahl method, as described by El-Shahaby [40] and Pirie [41], was used to calculate the total soluble nitrogen. The traditional semimicropropagation Kjeldahl method developed by Rees and Williams [42] and described by Haroun [43], was utilized to determine the total nitrogen. The amount of total soluble nitrogen was subtracted to determine the amount of insoluble protein.

Estimation of Total Protein
The content of protein was measured in accordance with Bradford [44] using spectrophotometry.

Chemical Constituents of Diethyl Ether Extract
Instrumentation 1 H NMR: The NMR spectra were recorded on a Varian Mercury VX-300 nmr spectrometer (Varian, Inc., Palo Alto, CA, USA). 1 H-NMR spectra were run at 300 MHz in deuterated chloroform (CDCl 3 ) or dimethyl sulphoxide (DMSO-d 6 ). Chemical shifts are quoted in δ relative to that of TMS.
GC/MS: The Finnigan SSQ 7000 Mass Selective Detector (MSD) interfaced with an Avarian GC was used to determine the MS identification of the GC components through the ICIS V2.0 data system. The DB-5 column (J & W Scientific, Folsom, CA, USA) utilized for this purpose was a 30-meter-long fused silica capillary with an internal diameter of 0.25 mm and a polydimethylsiloxane coating at a film thickness of 0.5 µm. The oven temperature was initially set to 50 • C and maintained isothermally for 3 min before being heated at a rate of 7 • C per minute to a final temperature of 250 • C, at which it was maintained isothermally for 10 min. The injector temperature was set to 200 • C and the volume injected was 0.5 µL. The transition line and ion source temperature were 250 • C and 150 • C, respectively. The mass spectrometer scanned the range of m/z 50 to m/z 300 with an ionization energy of 70 ev and a delay of 3 min to avoid the solvent pea (National Research Center (NRC), Dokki, Cairo, Egypt).
Processing of total diethyl ether extract (E) of A. subolivaceous/A. nilotica metabolite: The GC/MS technique was utilized to identify and characterize the components present in the total diethyl ether (E) extract sample.

Changes in Yield Attributes
The data shown in Figures 1 and 2 demonstrated that plants under drought stress treated with water and aqueous extracts showed a significant increase in shoot length by 10.78% and 10.06%, respectively; shoot fresh weight by 38.81% and 10.28%, respectively; shoot dry weight by 23.59% and 34.30%, respectively; legumes fresh weight by 132.75%, and 72.34%, respectively; seeds fresh weight by 150.70% and 120.41%, respectively; seeds dry weight by 176.63% and 118.55%, respectively; relative grain yield by 163.51% and 124.72%, respectively; and seed index by 12.41% and 11.98%, respectively, compared to control plants. Whereas drought-stressed plants treated with diethyl ether extract and ethyl acetate extract showed a significant increase in shoot length by 4.69% and 7.52%, respectively; shoot fresh weight by 20.93% and 14.64%, respectively; legumes fresh weight by 47.07% and 32.87%; respectively; seeds fresh weight by 52.18% and 46.34%, respectively; seeds dry weight by 61.69% and 45.06%, respectively; relative grain yield by 78.50% and 45.11%, respectively; and seed index by 17.26% and 10.81%, respectively, compared to control plants.   Figure 3 showed that A. subolivaceus water extracts (100% and 50%) led to a generally significant decrease in sucrose content of white lupine yielded seeds by 0.47% and 0.73%, respectively, and total soluble sugars content by 1.29% and 1.49%, respectively, while polysaccharides increased insignificantly by 5.60% and 0.61%, respectively. In this connection, water extract 50% decreased glucose and total carbohydrate content insignificantly by 2.47% and 0.56%, respectively, water extract 100% increased them insignificantly by 4.44% and 0.35%, respectively. Furthermore, diethyl ether extract 100% decreased all carbohydrate fractions of lupine yielded seeds insignificantly. On the other hand, the only insignificant decrease with diethyl ether 50% treatment was recorded for total soluble sugar content (0.28%). However, ethyl acetate extracts (100% and 50%) increased carbohydrate fractions significantly, in general. With the exception of glucose content that increased, the other carbohydrate fractions' content showed a generally insignificant decrease with aqueous extracts (100% and 50%). On the whole, the diethyl ether extract 50% and ethyl acetate extracts (100% and 50%) caused the maximum carbohydrate fractions content.

Changes in Nitrogen Content
Data presented in Figure 4 showed that the pretreatment with water and diethyl ether extracts (100% and 50%) caused a generally significant increase in different nitrogen fractions except water extract 100%, which caused a decrease in ammonia and amide content of the yielded seed by 1.96% and 19.56%, respectively. In this connection, the general significant increments in different nitrogen fractions were recorded for ethyl acetate 50% extract and aqueous extracts (100% and 50%), with the exception of a general significant decrease that was reported with those mentioned extracts for amide content, and with aqueous extract 100% for ammonia of lupine yielded seeds. On the other hand, ethyl acetate extract 100% caused an insignificant decrease in ammonia and amino nitrogen contents by 5.18% and 9.41%, respectively, a significant decrease in amide and total soluble nitrogen contents by 3.96% and 14.29%, respectively, and a significant increase in nitrogen, protein and total nitrogen of white lupine yielded seeds by 204.50% and 66.67%, respectively. However, the treatments that led to the maximum nitrogen content were recorded for water and diethyl ether extracts.

Changes in Protein Content
The obtained results showed that all treatments increased the protein content of the yielded Lupinus seeds significantly, except water extract 50%, which decreased it insignificantly by 0.76% ( Figure 5). Moreover, the pretreatment of seeds with diethyl ether extract 50% caused the maximum protein content above than control by 6.40%.

Processing of Total Diethyl Ether Extract (E) of A. subolivaceous/A. nilotica Metabolite
In order to explore the components of A. subolivaceous metabolites, the fungal metabolites produced by A. subolivaceous growth on powdered A. nilotica leaves were processed in a variety of ways (see Scheme 1). The extract's constituents were then identified using Wiley 9, Wiley 7, NIST, and main lib libraries, or by comparing literature data [45] to mass spectral databases after fractionation of the A. subolivaceous metabolite extract using various solvents, separation of its components, and analysis using 1 H-NMR spectra, and GC/MS. The pretreatment of L. termis seeds with diethyl ether extract showed the best results as caused the maximum increase in (glucose, amino nitrogen, insoluble protein nitrogen, total nitrogen, and protein) contents in the yielded seeds, so the diethyl ether extract was processed in order to study its components. It was found that A. subolivaceous metabolite extract is rich in phytochemicals such as fatty materials, terpenes, flavonoids, and other minor phytochemicals as shown in Table 2 and Figures 6 and 7.    In this investigation, the GC/MS analysis of diethyl ether extract from A. subolivaceous metabolite showed the presence of 4-methyl-p-menth-8-en-3-one and 3,4,7-trimethoxyquercetinas, shown in Table 3 and Figure 7, which have not reported previously from A. nilotica leaves extract as shown in Table 1, indicating the probable biotransformation as a result of the presence of A. subolivaceous. However, by a single or multiple step O-methylation process, A. subolivaceous can convert quercetin into 3 ,4 ,7 -trimethoxyquercetin. Additionally, A. subolivaceous has the ability to convert citronellal by one or more one-step C-methylation processes into 4-methyl-p-menth-8-en-3-one.

Changes in Yield Attributes
Concerning this, Khalil and Ismael [46] reported that the yeast application in the L. termis L. yielded seeds enhanced yield attributes (number of pods/plants, number of seeds/plants, and 100 seeds weight). Anwer and Khan [47], who showed that the nursery application of A. niger isolates considerably affected yield and tomato plants highest with maximal growth, corroborated the observed results. A. niger also typically increases crop production, nitrogen uptake, and root growth and development [48].
These findings are also in line with those of El-Shahawy et al. [49], who revealed that the use of yeast as a fungal application increased the yield attributes of Linum usitatissimum L. plants, including total plant length, straw yield per plant, number of fruiting branches per plant, number of capsules per plant, the weight of 1000 seeds, biological yield per plant, and seed yield per plant. Connectively, the use of biofertilizer boosted the fennel (Foeniculum vulgare Mill.) plants' yield features (plant fresh weight, plant dry weight, plant height, branches number, and fruit yield/plant) [50].

Changes in Carbohydrate Content
Application of A. subolivaceus extract resulted in a rise in the produced seeds' carbohydrate content, according to research on the influence of A. subolivaceus extracts on that fraction's content. However, the ethyl acetate (100% and 50%) and diethyl ether (50%) extracts produced the seeds with the highest carbohydrate fraction content. These findings are consistent with those of Khalil and Ismail [46], who observed an increase in the percentage of carbohydrates as a result of the use of yeast and attributed this increase to either an increase in chlorophyll a and b or to yeast's enhanced role in cell division and cell elongation, which resulted in more leaf area [51]. Mahfouz and Sharaf-Eldin [51] investigated the biochemical features of fennel seeds and found that application of biofertilizers increased total carbs content. The availability of inorganic nitrogen in particular may have an impact on how proteins, soluble solids, and secondary metabolites are synthesized [52]. A considerable amount of variance was seen in the level of plant metabolites in seeds among the six examined mung bean types, and they also reported a larger buildup of amino acid content in leaves.

Changes in Nitrogen Content
The recorded increase in nitrogen content was found to be consistent with Gomaa and Abou-Aly's findings [53], who investigated how yeast foliar treatment affected L. termis L. seeds and concluded that the yeast-produced growth hormones were considered to be the cause of the rise in nitrogen and protein content. El-Sayed et al. [54] further hypothesized that the rise in total nitrogen in the anise plants treated with bio-fertilizers may be the result of inoculation with non-symbiotic nitrogen-fixing organisms. Growth, which is impacted by several internal and external factors, in addition to its genetic make-up, is thus a valuable tool for assessing agricultural production across numerous crops. As a result, among other things, several factors, including water conductivity, osmotic potential, and threshold turgor, are essential components for plant growth and must all be present in order for growth to occur [55]. Nitrogen is an essential element of several components, such as proteins, enzymes, alkaloids, hormones, and vitamins [56]. It plays a significant role in photosynthesis process and the transfer of nutrients to the seed.

Changes in Protein Content
The observed increase in protein content was consistent with the results of Nafie [57], who reported that treatment with Streptomyces chibaensis increased protein content and biochemical components in L. termis L. seeds. Nonetheless, the same author also observed a decrease in protein content in L. termis L. seeds following treatment with Fusarium oxysporum, leading some to speculate that the plant's relatively low protein content was due to a high concentration of soluble sugars. The protein content is closely correlated with the nitrogen content [58], the tuber's protein content is crucial in terms of quality in potato tubers. The 100% manure × (Azospirillum + Pseudomonas) mixture had the highest tuber protein content (7.05%). By reactivating minerals found in soil and manure, the use of biofertilizers alongside manure to potatoes promotes the uptake of minerals from the soil and organic sources and decreases the demand for chemical fertilizers [59]. Application of both biofertilizer and other nitrogen fertilizers gave the high protein content of potato tuber [59,60].

Processing of Total Diethyl Ether Extract (E) of A. subolivaceous/A. nilotica Metabolite
The extract of the metabolite was discovered to be highly phytochemical. According to Kim et al. [61], A. subolivaceous, has various components, including sugars such as glucose, glucopyranose, galactose, turanose, and arabinofuranose, as well as sugar alcohols such as glycerol and inositol, organic acids such as citric acid and pyruvic acid, and fatty acids such as linoleic acid and palmitic acid. The current findings agreed with those of Senthilkumar et al. [62] and Devi and Prabakaran [63] who claimed that the metabolites generated by endophytic fungi Phomopsis sp. contained alkaloids, terpenoids, phenolics, and minor secondary compounds.
Devi and Prabakaran also studied the GC chromatogram and spectral analysis, as well as the name, molecular weight, and chemical structure of the compounds present in the ethyl acetate extract for Penicillium sp. These compounds include 4-hydroxybenzeneethanol, 2-tert-butyl-4-isopropyl-1-5-methyl phenol, and benzoic acid. 4-hydroxypropyl ester, p-hydroxyphenylacetamide, N-[2-methyl-1-prenylpropyl] formamide, cyclo (L-leucyl-Lpropyl), 3-(3-azidopropyl)-1H-indene, and dihydroergotamine are some examples of these compounds. However, Waqas et al. [64] investigated the potential of two endophytic fungi to release the phytohormones gibberellins (GAs) and indoleacetic acid (IAA). They claimed that the study of these endophytic fungi's pure cultures revealed varying levels of biologically active GAs (GA1, GA3, GA4, and GA7). The volatile compounds found in the culture samples of Trichoderma harzianum are members of the compound classes of alkanes, ketones, alcohols, pyrones (lactones), monoterpenes, furanes and sesquiterpenes [65]. A potent method for changing the structural makeup of bioactive natural and manmade materials is microbial transformation. The spectral analysis of the current study indicated biotransformation to a flavonoid (quercetin 7,3,4 trimethoxy) and a monoterpene (4-methyl P-menth-8-en-3-one), suggesting that biotransformation may be effective. The microorganism's O-methylation of the flavonols quercetin and fisetin, which takes place at the C-3 and C-4 hydroxyls, supports this conclusion [6]. In this regard, quercetin was oxidized enzymatically by A. flavus, and quercetin was then broken down by quercetinase to produce carbon monoxide and 2-protocatechuoylphloroglucinol carboxylic acid [3]. O-methylated quercetin was found in Streptomyces griseus (ATCC 13273) [66]. Quercetin's OH at C-3 can be methylated by Beauveria bassiana ATCC 7159 to produce 3 -O-methylquercetin, and these bacteria can also produce derivatives of 3 -O-methylquercetin-7-glucuronide by glycosylating the hydroxyl at C-7 of quercetin [67]. Additionally, quercetin was transformed into a variety of metabolites through the processes of hydroxylation, glucosylation, and methylation by species of Bacillus, Aspergillus, Streptomyces, and Penicillium. Similar to this, Rueda et al. [5] investigated the biotransformation of (S)-citronellal using Penicillium and Fusarium sp. to produce a number of compounds. This outcome is also consistent with the monoterpene (tetra hydrogeraniol) to hydroxycitronellol conversion described for Glomerella cingulate [68]. In this regard, Rhodotorulaminuta free and immobilized cells were used in the biotransformation of (L)-citronellol to (L)-citronellol by Velankar and Heble [69]. Additionally, Pulicaria undulata plants were regarded by Alshammari et al. [70] as a useful source of physiologically active secondary metabolites.

Conclusions
Lupinus seed has an important place in the pharmaceutical industry, and has been used for the production of protein isolates with good functional and nutritional properties that are considered a protein source for human nutrition and animal feeding all over the world. According to this study's findings, the fungal metabolite produced by A. subolivaceus growing on A. nilotica, is considered as an important strategy in agricultural sustainability. Hence, we must lessen our reliance on artificial fertilizers, which have detrimental effects on both the environment and human health, by using these naturally occurring substances to promote plant growth. This may lead to the development of a more ecologically conscious and economically viable farming system. It became clear that the bioactive natural and manufactured materials can have their structures changed effectively by microbial transformation as water and ether extracts improved the plant's growth and yield and biochemical characteristics of the produced Lupine seeds. The spectral analysis of the present study revealed biotransformation to a flavonoid (3 ,4 ,7 -trimethoxyquercetin) and a monoterpene (4-methyl P-menth-8-en-3-one), as well as maximization of the active compound production that is used as a source of medicinal materials in the pharmaceutical industry.