A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

doi:10.21203/rs.3.rs-4358366/v1

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A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

https://doi.org/10.21203/rs.3.rs-4358366/v1

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published 07 Aug, 2024

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Viola odorata plant extracts are extensively used in indigenous medicine to treat infectious diseases. V. odorata's limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. In particular, basal medium, carbon and nitrogen source, inoculum density, macronutrient concentration, shaking speed, temperature, pH, light intensity, and plant growth regulators were systematically optimized in order to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L^-1 to 21.68 ± 0.82 g DW L^-1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors, namely, stirred tank, airlift, and bubble column, to cultivate V. odorata biomass. Of these three methods, cultivation in a stirred tank reactor produced ~19.7 g DW L^-1 of maximum biomass within a 12-day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry based metabolomics, yielding around 18 key principles (metabolites) that exhibit resemblance with that in the natural source. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.

Viola odorata

bioprocess

optimization

bioreactor

metabolomics

antibacterial activity

antioxidant activity

Bioreactor cultivation of V. odorata plant cells with biomass productivity equivalent to that in shake flask culture, yielding extracts with a similar metabolite profile and associated bioactivity to that in the natural plant extracts.

The majority of pharmaceutical compounds originate from plants. Herbal extracts have been extensively used in traditional medicine to control and prevent diseases. According to recent World Health Organization (WHO) assessments, 80% of people globally depend on herbal medicines for their primary healthcare needs (WHO, 2023). Natural plant sources are limited in availability and the commercial demand for natural products has required a continuous supply of quality plant raw materials in reasonably adequate quantities. Plant cell bioprocessing (PCB) of in vitro plant cell and organ cultures is a promising alternative to natural plant biomass. It enables the continuous and sustainable production of therapeutically active plant biomass under controlled conditions (Georgiev, 2014). Thus, large-scale production of plant cells and organ cultures is possible in bioreactors with controllable production rates of desired phytochemicals (Shohael et al. 2014), thereby reducing the downstream processing cost of the desired metabolites in comparison to that obtained from the natural plants.

In vitro plant cell cultures are amenable to process optimization and scale-up for enhanced yield and productivity of their phytoconstituents (Shohael et al. 2006), with no regulatory barriers, generally associated with field-grown plants (Sparrow et al. 2007). Added merits of the system include the feasibility of cryopreservation (Park et al. 1998) to conserve the high-yielding cell lines for a sustained future with enhanced production. From a bioactivity perspective, in vitro cultures can effectively produce novel phytocompounds that are otherwise not detectable in the extracts of natural plants (Narayani et al. 2017a). From an ecological perspective, in vitro, plant cell cultivation conserves natural plants, additionally ensuring uniform and consistent quality and quantity of their natural products.

Viola odorata L. (known as 'Banafsha') is found in Kashmir and the western Himalayan regions of India at altitudes of 5000–6000 feet (Singh 1965). It has short stems (5–8 sm), heart-shaped leaves, and dark-purple flowers (Akhbari et al. 2012). It is widely used in Ayurveda, Siddha, Homoeopathy and Unani medicinal systems for the treatment of a variety of ailments like tonsillitis, respiratory catarrh, cough, bronchitis, asthma, rheumatism, malaria, and urinary disorders (Singh 1965; Koochek et al. 2003; Chandra et al. 2015). V. odorata extract has been meticulously reported for hepatoprotective, anticancer, cytotoxic, anti-tubercular, sedative, diuretic, anti-asthmatic, laxative, anti-dyslipidemia, antihypertensive, antibacterial, antidiabetic, antimicrobial, anti-inflammatory, antioxidant, antipyretic, and antifungal activities (Feyzabadi et al. 2017; Fazeenah and Quamri 2021). However, there is no study till date reporting the anti-microbial nature of V. odorata biomass extracts cultivated in bioreactors as reported in the current study.

On a commercial note, extracts of V. odorata is widely used as an effective ingredient in numerous natural products industries. For instance, a commercially available new herbal drug called ‘odorata’ has been formulated with active constituents from V. odorata (Mokhtari et al. 2015). V. odorata and its in vitro cultures are rich in cyclotides, a unique class of plant-based cyclic peptides, demonstrating many biological activities, including cytotoxic, anti-inflammatory and anti-bacterial activities (Narayani et al. 2020). In 2017, ‘Sero-X’, a bioinsecticide product, based on cyclotide extracts of Clitorea ternatea, was isolated and used on cotton plants to control insects including Helicoverpa armigera, H. punctigera, Bemisia tabaci and Creontiades dilutus. Interestingly, this bioinsecticide does not affect beneficial insects like bees (Parsley et al. 2018), thereby contributing to ecological balance. In South Africa, V. odorata oils combined with Santalum austrocaledonicum essential oils have been reported to show promising antibacterial activity with synergistic interaction (Orchard et al. 2023). Hence, evaluation of anti-microbial nature of bioprocessed plant biomass would be important to extend their commercial scope as potential products.

Though all the above-mentioned reports clearly indicate the commercial demand for V. odorata plant biomass, the plant is in danger of extinction in certain parts of India, due to its extensive wildcrafting (Malik et al. 2011). Besides, the seed germination rate in V. odorata is low due to seed dormancy (Barekat et al. 2013). Hence, large-scale plant cell culture-based cultivation of V. odorata would be an excellent alternative for natural plant biomass. On the other hand, the limitations associated with in vitro cultivation of plant cells include relatively slow growth compared to other production platforms, such as microbial systems, and a low yield of single-purified products (Chattopadhyay et al. 2002). However, these limitations could be overcome by applying suitable bioprocess strategies with robust design and implementation (Motolinía-Alcántara et al. 2021; Georgiev et al. 2018; Georgiev et al. 2009). The present study shows one such approach encompassing the application of OFAT and statistical optimization tools to achieve the optimal growth conditions in shake flasks, followed by implementing them in a bioreactor scale, resulting in enhanced V. odorata biomass production under controlled conditions.

Natural product industries have been using whole plant extracts, as prescribed in traditional medicinal systems, to bring desired synergistic effects based on their bioactivity, with an added merit of reduction in metabolite loss during purification. Moreover, under in vitro conditions, the phytochemical composition of the bioprocessed plant materials could be efficiently improved. In recent years, the successful establishment of in vitro cultures and laboratory-scale bioreactor cultivation of various plant species have been reported as follows:

Salvia viridis L (Grzegorczyk-Karolak et al. 2022), Ocinum basilicum (Pandey et al. 2019), Buddleja cordata (Vazquez-Marquez et al. 2019), Catharanthus roseus (Thakore et al. 2017), Stevia rebaudiana B (Vives et al. 2017), Rhododendron tomentosum (Jesionek et al. 2017), Scrophularia striata (Ahmadi-Sakha et al. 2016), Satureja khuzistanica (Khojasteh et al. 2016), Withania somnifera L. (Sivanandhan et al. 2014), and Aloe barbadensis (Mariateresa et al. 2014). However, this is the first report based on PCB of V. odorata under in vitro conditions.

In vitro cultures of V. odorata were earlier developed by Narayani et al. (2017a), wherein the cell and organ cultures of their extracts demonstrated a secondary metabolite profile similar to natural plants, including the cyclotides. (Narayani et al. 2017a, b). Also, V. odorata somatic embryo extracts were reported as non-haemolytic (HD50 = 756.7 µg ml^− 1) by Narayani et al. (2018), and it can be a promising lead for its application as a herbal drug formulation. For instance, a cough syrup prepared from V. odorata flowers effectively reduced the cough in children with asthma (Qasemzadeh et al. 2015). Classical and modern literature has proved that

V. odorata plant biomass extract rich in cyclotides could efficiently prevent and treat respiratory tract diseases and disorders, thereby motivating to test their anti-microbial nature in bacteria causing such respiratory tract disorders.

The current study intends to maximise the biomass yield (g DW of biomass. g of substrate^− 1) and productivity (g DW L^− 1 d^− 1) of the cell suspension culture of V. odorata using multi-factor optimization methodology (via the statistical design of experiments), taking into account the interactive effect of various physical and chemical parameters (Srivastava and Srivastava 2012a). In addition, the characterization and identification of key bioactive principles (phytochemicals) of in vitro culture extracts were thoroughly analysed using metabolomics based on chromatographic and mass spectrometric methods adapted from the literature (Narayani et al. 2017b). Antibacterial activity of in vitro-produced plant biomass extracts was tested against the bacterial strains causing respiratory tract infections with protocols reported earlier (Narayani et al. 2018). The antioxidant activity of in vitro-produced V. odorata plant biomass extracts was also studied and compared with natural plant biomass extract and vitamin E.

2.1 Developing cell suspension culture of V. odorata

V. odorata VOP-4 cells previously established in the Plant Cell Technology lab at the Indian Institute of Technology Madras (IITM) were maintained via periodic sub-culturing, as reported by Narayani et al. (2017a).

The pre-inoculum was prepared by suspending the callus (~ 6 g DW L^− 1) in 250 mL Erlenmeyer flasks containing 50 mL of woody plant medium (WPM) added with 3% (w/v) sucrose and 3 mg L^− 1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The cell suspension was then incubated in a shaker at 85 rpm with 16/8 h light/dark conditions at 23°C. After 14 days of biomass growth, the cell suspension was passed through a Buchner funnel to remove the large cell aggregates, while the harvested fine cells were used as inoculum (~ 6 g DW L^− 1) for further experiments.

2.2 Growth kinetics of V. odorata cell suspension culture

Knowledge of biomass production kinetics of V. odorata suspension culture was required to optimize various physical, chemical, and environmental parameters. For this, V. odorata cell suspension was grown in WPM with 3% (w/v) sucrose and 3 mg L^− 1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The initial pH was set at 5.7. The experiments were performed with an inoculum density of ~ 6 g DW L^− 1. V. odorata cells inoculated in 20 mL of medium in 100 mL Erlenmeyer flasks. The shake flasks were then kept in a shaker at 85 rpm and 16/8 h light/dark conditions at 23°C. The flasks were harvested (in triplicates) at regular intervals of 2 days until 20 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1). The biomass was freeze-dried to obtain the gram dry weight (g DW) and stored at -80°C for subsequent analysis. The specific growth rate and doubling time were calculated using the protocol reported by Srivastava and Srivastava (2012b).

2.3. Single and multifactor optimization of media components

2.3.1 Selection of plant basal medium for maximum biomass production in cell suspension culture of V. odorata

Basal salt formulations have a significant role in the growth of in vitro plant cell cultures. Hence, this study tested different basal plant growth media to maximize biomass production in V. odorata cell suspension culture. Murashige and Skoog modified medium (MMS) (Phillips and Garda 2019), Schenk & Hildebrandt medium (SH) (Schenk and Hildebrandt 1972), Murashige and Skoog medium (MS) (Murashige and Skoog 1962), Woody Plant medium (WPM) (Lloyd and McCown 1980), Gamborg B5 medium (B5), NB medium (NB) (Gamborg et al. 1968), Gresshoff &Doy medium (DBM2) (Kaul 1987), CHU N6 medium (CHU N6) (Chu et al. 1975), Nitsch medium (NM) (Nitsch and Nitsch 1969), White medium (White) (White 1954), Eriksson medium (ER) (Eriksson 1965) and Linsmaier & Skoog medium (LS) (Schumacher et al. 1987) were the different basal salt media used in this study. The experimental conditions were followed as mentioned in section 2.2.

V. odorata cells inoculated in 20 mL of medium in 100 mL Erlenmeyer flasks The flasks were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.3.2 Effect of carbon source on biomass production in cell suspension culture of V. odorata

Growth and metabolite production are highly dependent on the limiting nutrients present in the medium. Hence, to determine a suitable carbon source for maximum biomass production, V. odorata cell suspension was grown separately in a MS medium fortified with sucrose, glucose, and fructose (3%, w/v). The initial pH was set at 5.7. The experiments were performed with an inoculum density of ~ 6 g DW L^− 1. V. odorata cells inoculated in 20 mL of medium in 100 mL Erlenmeyer flasks. The shake flasks were then kept in a shaker at 85 rpm and 16/8 h light/dark conditions at 23°C. The flasks were harvested (in triplicates) at regular intervals of 2 days until 20 days of the cultivation period to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.3.3 Effect of inoculum density on biomass production in cell suspension culture of V. odorata

Previous reports from literature (Bansal et al. 2017; Zhang et al. 2019; Naik and Al-khayri 2020; Kumar et al. 2023) showed that the optimal inoculum density can lead to higher biomass concentration and yield with respect to substrate. Hence, experiments were performed at different inoculum densities ranging from 3 to 11 g DW L^− 1. V. odorata cells inoculated in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate the biomass yield using Eq. 1.

The yield of biomass to substrate (Y_x/s)\(=\frac{X-{X}_{0}}{{S}_{0}-S}\) (1)

where, X is the final biomass concentration (after 12 days), and X₀ is the initial biomass concentration (on day 0). S and S₀ are the final and initial substrate concentrations in the medium, respectively.

2.3.4 Effect of ammonium to nitrate ratio on biomass production in cell suspension culture of V. odorata

Ammonium and nitrate ions have different roles in plant metabolism (Li et al. 2013). The ammonium-to-nitrate ratios have been shown in literature to have an influence on biomass growth in plant cell cultures (Praveen et al. 2011). Hence, the effect of the ammonium-to-nitrate ratio (NH₄⁺:NO₃⁻ ) was studied in a varied range from 0:60 to 60:0. Ammonium chloride was used as an ammonium source, and potassium nitrate was used as the nitrate source. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The shake flasks (with culture broth) were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.3.5 Plackett–Burman design (PBD) to select the most significant nutrients

Plackett-Burman Design (PBD) experiments were used to select the medium nutrients that significantly affected biomass production in the cell suspension culture of V. odorata. This information can be instrumental in optimizing the medium composition to maximize biomass productivity.

PBD can be an effective and simple design of experiments for selecting essential factors that can affect biomass production. It is a tool that can be used to see the positive and negative effect, if any, of all the factors chosen (medium nutrients in this case) in the given concentration range on the response (biomass concentration in this case). Based on this information, the factors can be ranked and selected for further optimization using other designs like Response Surface Methodology (RSM) to maximize the response (biomass concentration). Based on previous experiments and literature reports, sucrose (A), ammonium chloride (B), calcium chloride dihydrate (C), magnesium sulfate hexahydrate (D), potassium nitrate (E), potassium phosphate monobasic (F), microelements (G), vitamins (H) and glycine(J) were the factors selected for PBD. Half and 2X MS concentrations (for high level, each factor was kept at 2X concentration; for low level, each factor was kept at 0.5X concentration) were kept at low and high level (Table 1), respectively. The design was developed using Design-Expert software (Stat-Ease Corporation, USA), version 11. Fifteen experiments were formulated using nine nutrients, and responses were measured in terms of biomass (g DW L^− 1) produced in each experimental recipe implemented. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

Table 1

Responses obtained from the experimental recipes of PB design to select the significant nutrients present in MS medium.
Run	A:Sucrose	B:Ammonium chloride	C:Calcium chloride dihydrate	D:Magnesium sulphate hexahydrate	E:Potassium nitrate	F:Potassium phosphate monobasic	G:Microelements	H:Vitamins	J:Glycine	K:Dummy 1	L:Dummy 2	Biomass
	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	X times	X times	mg L^− 1	mg L^− 1	mg L^− 1	g DW L^− 1
1	35000	1336.25	550	462.5	2375	212.5	1.25	1.25	2.5	0	0	14.9 ± 0.19
2	35000	1336.25	550	462.5	2375	212.5	1.25	1.25	2.5	0	0	14.4 ± 1.15
3	10000	2138	880	740	950	85	0.5	2	1	1	1	5.8 ± 0.11
4	60000	2138	220	185	950	340	0.5	2	4	-1	1	9.1 ± 0.61
5	10000	2138	880	185	3800	340	2	0.5	1	-1	1	5.6 ± 0.24
6	10000	534.5	220	185	950	85	0.5	0.5	1	-1	-1	6.1 ± 0.32
7	10000	2138	220	740	3800	85	2	2	4	-1	-1	5.1 ± 0.13
8	60000	534.5	220	185	3800	85	2	2	1	1	1	16 ± 1.14
9	10000	534.5	880	185	3800	340	0.5	2	4	1	-1	5.8 ± 0.13
10	60000	534.5	880	740	950	340	2	2	1	-1	-1	12.5 ± 0.28
11	60000	534.5	880	740	3800	85	0.5	0.5	4	-1	1	16 ± 1.26
12	60000	2138	880	185	950	85	2	0.5	4	1	-1	8.2 ± 0.40
13	10000	534.5	220	740	950	340	2	0.5	4	1	1	5.5 ± 0.16
14	35000	1336.25	550	462.5	2375	212.5	1.25	1.25	2.5	0	0	13.5 ± 0.40
15	60000	2138	220	740	3800	340	0.5	0.5	1	1	-1	8.9 ± 0.49

2.3.6 Determination of the optimum concentrations of the selected medium components using Response Surface Methodology (RSM)

Central Composite Designs (CCD) is an effective experimental design tool for optimizing the concentration of essential factors that affect biomass/metabolite production. It is a tool that can be used to analyze the interactive effect of the factors chosen (medium nutrients in this case) in the given concentration range on the desired response (biomass concentration in this case). According to PBD experiments (section 3.6), sucrose, ammonium chloride, and potassium nitrate are the medium nutrients that significantly influenced biomass production in the cell suspension culture of V. odorata. Hence, each nutrient was tested at full factorial points, centre points, and axial points (Table 2). The centre point was replicated six times to obtain five degrees of freedom for experimental error. The experimental design consisted of 20 runs (Table 2) for three independent variables, and each variable was tested at five concentration levels (Table 2). The experimental design (Table 2) was developed using Design-Expert software (Stat-Ease Corporation, USA), version 11.

Table 2

Responses obtained from the experimental recipes of central composite designs for optimization of medium composition.
Run	A: Sucrose (mg L^− 1)	B: Ammonium chloride (mg L^− 1)	C: Potassium nitrate (mg L^− 1)	Biomass (g DW L^− 1)
1	6364.1	685	1900	5.1 ± 0.01
2	40000	685	1900	18.3 ± 0.13
3	40000	1332.5	1900	12.8 ± 0.49
4	40000	685	1900	16.6 ± 1.36
5	20000	1070	950	7.5 ± 0.03
6	40000	37.5	1900	16.5 ± 0.64
7	20000	300	2850	11.3 ± 0.02
8	60000	300	950	18.2 ± 0.35
9	20000	1070	2850	9.5 ± 0.01
10	40000	685	1900	18.2 ± 0.47
11	40000	685	3497.7	15.8 ± 0.51
12	60000	1070	950	8.1 ± 0.18
13	40000	685	1900	17 ± 1.23
14	20000	300	950	9 ± 0.00
15	73635.9	685	1900	17.6 ± 0.65
16	40000	685	1900	17.3 ± 0.12
17	40000	685	1900	17.5 ± 0.42
18	60000	300	2850	18.5 ± 1.09
19	60000	1070	2850	17.4 ± 1.48
20	40000	685	302.3	5.1 ± 0.24

Twenty experiments were formulated using three nutrients, and responses were measured in terms of biomass (g DW L^− 1) produced in each experimental recipe. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.4 Single and multifactor optimization of cultivation conditions

2.4.1 Effect of rpm on biomass production in cell suspension culture of V. odorata

The optimum shaking speed is required to adequately transfer nutrients from the culture medium to cells (Chattopadhyay et al. 2002). V. odorata cell suspension was grown separately at different rpm ranging from 65 to 165 in an optimized medium composition to determine a suitable shaking speed for maximum biomass production. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1). Morphological changes due to the shear force in the cell suspension were studied. For this, V. odorata cell suspension (10 µL) was pipetted into a glass plate and examined under a Nikon Eclipse 80i microscope with imaging software (NIS- Elements-NIKON). Also, the cell viability was estimated using the TTC assay protocol described in section 2.6.1.

2.4.2 Effect of temperature and pH on biomass production in cell suspension culture of V. odorata

Temperature and pH affect cell growth and metabolite production in plant cell culture (Chattopadhyay et al. 2002). CCD was used to determine the optimum temperature and pH for maximum biomass production. The experimental design shown in Table 3 was developed using Design-Expert software v12 (Stat-Ease Corporation, USA). Fourteen experiments were formulated using 5 levels of temperature and pH. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

Table 3

Responses obtained from the experimental recipes of central composite designs for temperature and pH optimization.
Run	A: Temperature (° C)	B: pH	Biomass (g DW L^− 1)
1	23	5.7	18.7 ± 0.6
2	29.4	5.7	19.2 ± 0.4
3	23	5.7	17.3 ± 0.8
4	23	4.3	19 ± 1.1
5	27.5	6.7	18.8 ± 0.4
6	18.5	4.7	8.9 ± 0.6
7	27.5	4.7	18.2 ± 0.2
8	16.6	5.7	7.6 ± 0.5
9	18.5	6.7	6.8 ± 1.1
10	23	5.7	18.5 ± 0.2
11	23	5.7	18.9 ± 0.1
12	23	5.7	18.5 ± 0.8
13	23	7.1	13.9 ± 0.2
14	23	5.7	18.7 ± 0.1

2.4.3 Effect of light intensity on biomass production in cell suspension culture of V. odorata

Light intensity significantly affects in vitro plant biomass and metabolite production (Arias et al. 2016; Andi et al. 2019). Hence, cells were cultured at different light intensities to determine a suitable light intensity for maximum biomass production in V. odorata plant cell cultivation, ranging from complete dark to 5200 Lux in an optimized medium composition. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The flasks were harvested (in triplicates) after 12 days, to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.4.4 Effect of plant growth regulators (PGR) on biomass production in cell suspension culture of V. odorata

Screening the plant growth regulators (PGRs) with a regular OFAT approach costs much time and involves numerous experimental trials. Statistical optimization tools are helpful in understanding the factors' main effects (PGRs) with minimal experimental runs. Fractional factorial design with resolution IV was used to screen the significant PGRs that improve biomass production. Different PGRs were tested at two concentration levels (0.1 and 0.5 mg L^− 1, Table 4). The following PGRs were used in the study: 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BAP) (Narayani et al. 2017a), Indole-3-acetic acid (IAA), Kinetin, Thidiazuron (TDZ), Gibberellic acid (GA3) (Muhammad et al. 2013), Indole-3-butyric acid (IBA) (Vasilev et al. 2013), Indole-3-propionic acid (IPA) (Pattnaik et al. 2000), Picloram (Chavan et al. 2011), 2-Napthoxyacetic Acid (NOA) (Meziani et al. 2015), Adenine sulphate (AdS) (Naaz et al. 2014), Putrescine dihydrochloride (PHC) (Keighobadi et al. 2020), and Phloroglucinol (PG) (da Silva et al. 2013). The design (shown in Table 4) was developed using Design-Expert software v12 (Stat-Ease Corporation, USA), and it included 32 design points and 3 centre points with 14 experimental variables (PGRs). The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The initial pH was set at 5.4. The shake flask cultures were incubated in an orbital shaker at 26.6°C, 85 rpm and 16/8 h light/ dark conditions. Shake flasks were harvested (filtered through filter paper under vacuum) after 12 days, and the generated biomass (g DW L^− 1) was measured.

Table 4

Responses obtained from experimental recipes of fractional factorial design to select the significant plant growth regulators.
Run	A:2,4-D	B: IAA	C: IBA	D:NAA	E:IPA	F:Picloram	G:NOA	H:AdS	J:BAP	K:Kinetin	L:TDZ	M:GA3	N:PHC	O:PG	Biomass
	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	g DW L^− 1
1	-1	-1	1	-1	-1	1	-1	1	1	-1	1	-1	1	-1	21.5 ± 0.52
2	1	1	1	-1	1	1	-1	-1	-1	1	1	-1	1	-1	20.55 ± 0.66
3	-1	1	-1	-1	1	1	1	-1	1	-1	1	1	-1	-1	20.06 ± 1.01
4	-1	-1	-1	1	-1	-1	1	1	1	-1	-1	1	-1	1	20.08 ± 0.62
5	1	-1	1	-1	-1	-1	1	-1	1	1	-1	1	1	-1	19.65 ± 0.08
6	1	-1	-1	1	1	1	-1	-1	1	-1	-1	1	1	-1	19.8 ± 0.52
7	0	0	0	0	0	0	0	0	0	0	0	0	0	0	21.76 ± 0.05
8	1	1	-1	1	-1	-1	1	-1	-1	-1	1	-1	1	-1	21.72 ± 0.09
9	-1	-1	1	1	1	1	1	-1	-1	1	-1	-1	-1	-1	21.81 ± 0.68
10	-1	-1	-1	-1	1	-1	-1	-1	-1	1	1	1	1	1	22.05 ± 0.15
11	1	-1	-1	-1	-1	1	1	1	-1	1	1	1	-1	-1	19.95 ± 0.62
12	1	1	-1	1	1	-1	1	-1	-1	1	-1	1	-1	1	20.2 ± 0.12
13	-1	1	1	1	-1	-1	-1	-1	1	1	1	1	-1	-1	21.85 ± 0.2
14	1	1	1	1	-1	1	1	1	1	-1	-1	-1	-1	-1	20.25 ± 0.06
15	1	-1	-1	-1	1	1	1	1	-1	-1	-1	-1	1	1	19.82 ± 0.72
16	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	21.68 ± 0.09
17	1	-1	-1	1	-1	1	-1	-1	1	1	1	-1	-1	1	20.78 ± 0.76
18	0	0	0	0	0	0	0	0	0	0	0	0	0	0	20.68 ± 0.51
Run	A:2,4-D	B: IAA	C: IBA	D:NAA	E:IPA	F:Picloram	G:NOA	H:AdS	J:BAP	K:Kinetin	L:TDZ	M:GA3	N:PHC	O:PG	Biomass
	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	mg L^− 1	g DW L^− 1
19	1	1	-1	-1	-1	-1	-1	1	1	-1	1	1	1	1	19.46 ± 0.05
20	1	-1	1	-1	1	-1	1	-1	1	-1	1	-1	-1	1	20.96 ± 0.14
21	-1	1	1	1	1	-1	-1	-1	1	-1	-1	-1	1	1	21.02 ± 0.01
22	1	1	-1	-1	1	-1	-1	1	1	1	-1	-1	-1	-1	21.11 ± 0.02
23	-1	1	-1	-1	-1	1	1	-1	1	1	-1	-1	1	1	21.22 ± 0.37
24	-1	1	1	-1	-1	-1	1	1	-1	1	1	-1	-1	1	22.07 ± 0.09
25	1	-1	1	1	-1	-1	-1	1	-1	1	-1	-1	1	1	20.79 ± 0.06
26	1	1	1	1	1	1	1	1	1	1	1	1	1	1	19.12 ± 0.63
27	1	1	1	-1	-1	1	-1	-1	-1	-1	-1	1	-1	1	19.43 ± 0.84
28	-1	1	-1	1	-1	1	-1	1	-1	1	-1	1	1	-1	20.37 ± 0.34
29	-1	-1	-1	1	1	-1	1	1	1	1	1	-1	1	-1	21.79 ± 0.67
30	1	-1	1	1	1	-1	-1	1	-1	-1	1	1	-1	-1	20.62 ± 0.26
31	-1	1	1	-1	1	-1	1	1	-1	-1	-1	1	1	-1	20.39 ± 0.03
32	-1	-1	1	-1	1	1	-1	1	1	1	-1	1	-1	1	17.65 ± 1.01
33	0	0	0	0	0	0	0	0	0	0	0	0	0	0	21.15 ± 0.27
34	-1	1	-1	1	1	1	-1	1	-1	-1	1	-1	-1	1	20.7 ± 0.82
35	-1	-1	1	1	-1	1	1	-1	-1	-1	1	1	1	1	20.04 ± 0.06
* In Table 4, the values are represented as follows: "-1" corresponds to 0.1 mg L^− 1, "0" corresponds to 0.3 mg L^− 1, and "1" corresponds to 0.5 mg L^− 1 of the respective PGRs.

2.4.5 Determination of the optimum concentrations of the selected PGR using response surface methodology (RSM)

CCD has been used to understand the interactive effect of the factors (PGR) chosen in the given concentration range on the desired response (biomass concentration in this case) in this study. TDZ and 2,4 – D are the PGRs that demonstrated a considerable impact on biomass production in the cell suspension culture of V. odorata, according to the fractional factorial design of experiments. These two PGRs were tested at full factorial points, centre points and axial points. The centre point is replicated six times to obtain five degrees of freedom for experimental error. The experimental design consists of 14 runs (as shown in Table 5) for two independent variables, and each variable was tested at four concentration levels. The experimental design shown in Table 5 was developed using Design-Expert software v12 (Stat-Ease Corporation, USA). Fourteen experiments were formulated using two PGRs, and responses were measured in terms of biomass (g DW L^− 1) produced in each experimental recipe. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The initial pH was set at 5.4. The shake flask cultures were incubated in an orbital shaker at 26.6°C, 85 rpm and 16/8 h light/ dark conditions. After 12 days, the flasks were harvested (in triplicates) to estimate the generated biomass (g DW L^− 1).

Table 5

Responses obtained from the experimental recipes of central composite designs for PGR optimization.
Run	A: 2,4 – D (mg L^− 1)	B: TDZ (mg L^− 1)	Biomass (g DW L^− 1)
1	0.05	0.55	21.9 ± 0.17
2	0.05	0.55	22 ± 0.71
3	0.05	0	17.8 ± 1.5
4	0.12	0.55	21.5 ± 1.07
5	0.1	0.1	19.8 ± 0.17
6	0.05	1.19	20.8 ± 0.44
7	0	0.1	16.7 ± 0.2
8	0	1	19.9 ± 0.71
9	0.1	1	22.5 ± 0.9
10	0.05	0.55	21.7 ± 0.76
11	0	0.55	21.1 ± 0.54
12	0.05	0.55	22.2 ± 0.74
13	0.05	0.55	20.9 ± 0.81
14	0.05	0.55	21 ± 1.2

2.4.6 Effect of antifoam on biomass production in cell suspension culture of V. odorata

A suitable antifoam concentration can control the foaming, but it must also be non-toxic to the plant cells (Chattopadhyay et al. 2002). Hence, to determine a suitable antifoam concentration for maximum cell viability, the V. odorata cell suspension was grown separately at different antifoam (Silicone antifoaming agent – Himedia India) concentrations ranging from 0.012–0.12% in the optimized medium composition. The experiments were performed by inoculating ~ 7 g DW L^− 1 of V. odorata cells in 20 mL of medium in 100 mL Erlenmeyer flasks. The initial pH was set at 5.4. The shake flask cultures were incubated in an orbital shaker at 26.6°C, 85 rpm and 16/8 h light/ dark conditions. After 12 days, the cells (from shake flasks) were harvested (in triplicates) for biomass (g DW L^− 1), and analysed for residual sucrose (g L^− 1) and viability of the cells.

2.5 Batch bioreactor cultivation of V. odorata plant cells

2.5.1 Stirred tank reactor (STR)

The successful cultivation of plant cell suspension culture in lab-scale batch bioreactors was reported for the production of various phytochemicals (Pandey et al. 2019; Macharoen et al. 2021). The shake flask optimized V. odorata cell suspension culture conditions were applied in bioreactors to scale up the cell suspension culture. The batch bioreactor cultivation of V. odorata plant cells was performed in a 3 L STR Applikon bioreactor with 2.4 L of media and inoculum density of ̴7 g DW L⁻¹. The initial pH was adjusted to 5.4 before autoclaving and allowed to vary during the cultivation. Marine impeller and stainless-steel sintered sparger were used for agitation and aeration. Initially, the agitation speed was kept at 85 rpm, and the aeration was at 0.1 vvm. The agitation and aeration rates were varied during the cultivation period to prevent biomass from settling at the bottom of the reactor and to maintain the dissolved oxygen > 20% saturation during the cultivation. The medium evaporation was reduced by circulating cold water (< 10°C) through the exit gas condenser. A light jacket (LED) was used to maintain a 16/8 h (light/dark) photoperiod (200 Lux), and the temperature of the reactor was maintained at 26.6°C by circulating the cold/warm water in the cooling finger. A silicone-based antifoam agent was used at a concentration of 0.012% (v/v) to control the foaming. After every 24 h, samples were taken from the bioreactor to estimate the generated biomass (g DW L⁻¹) and utilized substrate (g L⁻¹).

2.5.2 Airlift bioreactor (ALR)

Pneumatically driven bioreactors are the preferred choice for shear-sensitive cell cultures. The batch bioreactor cultivation of V. odorata plant cells was performed in a 3.5 L airlift bioreactor with 3 L of media and inoculum density of ̴ 3.5 g DW L⁻¹. The H: D ratio of the airlift reactor was kept at 4. The internal draft tube measured a height of 27 cm and a diameter of 5 cm. The initial pH was kept at 5.4. A stainless-steel ring sparger (diameter 3.5 cm) was used for aeration. The aeration rate was kept at 0.1 vvm. The aeration rate was varied during the cultivation period to ensure proper mixing and to avoid biomass settling at the base of the reactor and to maintain the dissolved oxygen > 20% saturation during the cultivation. A light jacket (LED) was used as a light source to maintain a 16/8 h light/dark photoperiod (200 Lux), and the temperature of the suspension culture was maintained at 26.6°C with a temperature controller. The cold water (< 10°C) was circulated in the cooling tube (U shape), and a heating rod was used to maintain the temperature. A silicone-based antifoam agent was used at a concentration of 0.012% (v/v) to control the foaming. From the fourth day onwards, after every 24 h, samples were withdrawn from the bioreactor to estimate the biomass concentration (g DW L⁻¹) and utilized substrate (g L^− 1).

2.5.3 Bubble column bioreactor (BCB)

A bubble column bioreactor (BCB) is simple to construct and provides a low-shear environment compared to STR. Phlojodicarpus sibiricus cell suspension cultures were cultivated in BCBs and showed better growth than STR (Khandy et al. 2021). The batch bioreactor cultivation of V. odorata plant cells was performed in a 3.5 L bubble column bioreactor with 3 L of medium and inoculum density of ̴ 3.5 g DW L⁻¹. The dimensions were similar to that of the airlift reactor used previously. The initial pH was kept at 5.4. A stainless-steel ring sparger was used for aeration. The initial aeration rate was kept at 0.1 vvm. The aeration rate was varied during the cultivation period to ensure proper mixing and avoid biomass settling and to maintain the dissolved oxygen > 20% saturation during the cultivation. A light jacket (LED) source was used to maintain a 16/8 h light/dark photoperiod (200 Lux), and the temperature was maintained at 26.6°C with a temperature controller. The cold water from the chilling unit (~ 10°C) was circulated in the cooling tube (U shape), and a heating rod was used to maintain the temperature. A silicone-based antifoam agent was used at a concentration of 0.012% (v/v) to control the foaming. From the third day onwards, every 24 hours, samples were taken from the bioreactor to estimate the generated biomass (g DW L^− 1) and utilized substrate (g L^− 1).

2.6 Analytical methods

2.6.1 Cell viability assay

The effect of rpm and antifoam on cell viability was estimated with the Tetrazolium trichloride (TTC) assay. The protocol reported by Towill and Mazur (1975) was followed with minor modifications. 10 mg of fresh V. odorata cells were treated with 3 mL TTC solution and incubated for 18 h at 25°C. The samples were heated for 15 min at 60°C. The formazan was extracted in ethanol, and the optical density was measured at 485 nm. The viability of V. odorata cells was calculated, as reported earlier by Srikantan et al. (2018).

2.6.2 Estimation of residual carbon source using high-performance liquid chromatography (HPLC)

Residual carbon was estimated using a high-performance liquid chromatography (HPLC) protocol reported by Srikantan et al. (2018). Briefly, the samples were filtered (0.2 µm filters) and analyzed using HPLC (Agilent Technologies, USA). Water was used as the mobile phase at the flow rate of 0.6 ml min^− 1, and an RCM monosaccharide column (Phenomenex, USA) was used as the stationary phase. The column oven temperature was maintained at 80°C. The standard plots of sucrose, glucose, and fructose were used to calculate the residual carbon (g L^− 1) present in the samples.

2.6.3 Estimation of total flavonoid and total phenolics

Plant secondary metabolites are reported for their various biological activities. The metabolites like eugenol, phytol, tetradecanoic acid, hexadecanoic acid, methyl ester, and octacosanol have been reported for numerous biological activities such as anti-tumour necrosis factor, antioxidant, antiseptic, anti-inflammatory, antidiabetic and antimicrobial activities. Along with cyclotides, as reported in the plant cell culture of V. odorata (Narayani et al. 2017b), these phytochemicals can contribute to several bioactivities of V. odorata. Hence, this study measured the total phenolic and flavonoid contents in V. odorata plant biomass. The plant biomass was extracted according to the protocol reported by Fulzele and Satdive (2005). Total phenolic content (TPC) was analyzed using the Folin–Ciocalteu method (Folin and Ciocalteau 1927), and the total flavonoid content (TFC) was determined using the method described by Wu et al. (2006).

2.6.4 GCMS and LCMS analysis of V. odorata plant biomass

V. odorata plant materials were extracted using the protocol reported by Narayani et al. (2017b). The powdered plant biomass extract was dissolved (10 mg mL^− 1) in methanol and analyzed with the GCMS protocol reported earlier by Zhang et al. (2013). The DB-5 MS fused-silica capillary column (length 30 m, I.D 0.25 mm) was used. Electron ionization was done at 70 eV. The oven operated isothermally, and the temperature was maintained at 70°C for 4 min. Then, the temperature was raised at 5°C min^− 1 up to 310°C and held for 10 min. The sample injection temperature was 290°C. The carrier gas (Helium) flow rate was 1 mL min^− 1, and the 1 µL sample was injected with a split ratio of 1/10. The ion source and transfer line temperatures were 230°C and 280°C, respectively. The mass spectra plot was acquired using full scan monitoring mode with a mass scan range of m/z 50 − 600, and solvent delay of 8 min.

Narayani et al. (2017a) reported the protocol for cyclotide analysis using LC-FTMS. The samples were analyzed in an Orbitrap Elite mass spectrometer coupled to an ESAY – nLC™ liquid chromatography unit via a Nano electrospray ion source equipped with a stainless-steel nano-emitter. The chromatography operated with a binary solvent system. Solvent A – water/acetonitrile (95:5 v/v) with 0.1% (v/v) formic acid and Solvent B - acetonitrile/water (95:5 v/v) with 0.1% (v/v) formic acid. Samples were separated by gradient mode with a 300 nL min^− 1 flow rate. The samples were loaded from a 96-well microplate autosampler by using an EASY- nLC system at 3 µL min^− 1. The samples were concentrated on a trapping column (Easy-column™, L 2 cm, ID 100 µM, 120 A°, C18-A1) and were eluted and directed onto a reverse phase column (Easy-column™, L 10 cm, ID 75 µM, 120 A°, C18-A2 ). The instrument operated at a higher resolution (120000) in data-dependent mode, automatically switching between MS and MS/MS. The mass scan range was kept as 500–2000. The monoisotopic mass of cyclotides was manually verified by directly calculating the peak positions and charge states using Xcalibur Qual Browser.

2.7 Bioactivity analysis of V. odorata plant biomass extracts

2.7.1 Antimicrobial activity of V. odorata plant biomass extract

As reported earlier, the protocol for cultivating natural plants and in vitro cultures was followed (Narayani et al. 2017a; 2018). This study used the following V. odorata plant materials: D1: natural plant, D2: in vitro plant, D3: somatic embryos, D4: STR cultivated cell suspension, and D5: ALR cultivated cell suspension biomass extracts. The harvested plant biomass was extracted using the protocol reported by Narayani et al. (2017b).

The antibacterial activity of the V. odorata cell culture extracts and the natural plant was tested against respiratory tract pathogens using a resazurin microtiter assay protocol reported by Sarker et al. (2007). In brief, Muller Hinton broth medium was inoculated with selected bacterial strains (procured from the Microbial Type Culture Collection (MTCC) IMTECH, Chandigarh, India) known to cause respiratory tract infections (MTCC 442 - Streptococcus pyogenes, MTCC 655- Streptococcus pneumoniae, and MTCC 3826 Haemophilus influenzae). The plant biomass extract (200 to 1.56 mg L^− 1) was mixed with bacterial culture (inoculated at 0.5 McFarland turbidity standard), and bacterial growth was assessed with a resazurin indicator solution. The microbial growth was observed visually (change of colour from blue to pink). The plant extract concentration at which the colour change was prevented was noted as Minimum Inhibitory Concentration (MIC) for the corresponding tested microorganism. The MIC was defined as the lowest concentration of each test compound or drug that prevented any colour change (observed visually), i.e. which completely inhibited microbial growth.

2.7.2 Antioxidant activity of V. odorata plant biomass extract

Free radical molecule leads to the oxidation of genetic and protein molecules, resulting in cell damage and death (Ahmadinejad et al 2017). Moreover, free radicals induce cancer and heart diseases (Ahmadinejad et al 2017). Antioxidant molecules protect the biomolecules from free radicals (Stojković et al. 2011). Hence, the antioxidant activity of the V. odorata biomass extracts (prepared as described in section 2.7.1) was tested by the DPPH (2, 2’-Diphenyl-1-picrylhydrazyl)) assay reported in the literature (Tanaka et al. 2003). The DPPH scavenging potential of the samples was calculated as follows.

\(\% DPPH radical scavenging activity=\frac{Control OD-Sample OD}{Control OD}\) ⨯100

2.8 Statistical analysis

All the studies were done in triplicates, excluding bioreactor cultivations, and the data were represented as mean ± standard deviation (SD). The calculated means from OFAT experiments were analysed in a one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. The mean difference was considered statistically significant when p < 0.05. Statistical analysis was performed using GraphPad Prism (v.9.5.1 - GraphPad Software Inc). The statistical experimental designs were developed and analysed using Design-Expert software v12(Stat-Ease Corporation, USA).

3.1 Growth kinetics of V. odorata cell suspension culture

To develop the batch kinetics with the established cultivation protocol for V. odorata (Narayani et al. 2017a), the growth curve of the V. odorata cell suspension was plotted over a three-weeks time frame (Fig. 1a). Maximum biomass (10.2 ± 0.8 g DW L^− 1) was obtained on the 12th day, with a residual sucrose concentration of 0.12 g L^− 1. Hence, 12 days of cultivation period was used as batch-time for further studies (OFAT). The specific growth rate of the V. odorata cell suspension culture was estimated to be 0.074 d^− 1. Changes in electrical conductivity and medium pH were also recorded over time. A decrease in the medium conductivity from 2326 µS to 1926 µS reflects on the uptake of salts from the medium for growth. However, no significant change in pH could be observed from the initial value of 5.7. The sugar analysis in the medium demonstrated that sucrose present in the medium was rapidly converted into glucose and fructose during the early phase of growth, as evidenced by the increase in glucose and fructose concentration in the medium (Fig. 1a), which subsequently got consumed during the active phase of growth. The initial increase in the concentration of monosaccharides (glucose and fructose) with a rapid reduction in sucrose was possibly due to the plant cell wall invertases (Kretzschmar et al. 2007). Interestingly, the glucose consumption rate exceeded fructose during the culture growth. Hence, another experiment was designed to compare the biomass productivity and yield with respect to a substrate using glucose as a sole carbon source, keeping other cultivation conditions the same (using OFAT approach), the biomass concentration of 10.1 ± 1.1 g DW L^− 1 with residual glucose of 17.4 g L^− 1 was obtained after 12 days. The medium pH varied in the range of 5.3 to 5.6. Thus, glucose as a carbon source could result in biomass productivity of 0.72 g DW L^− 1 d^− 1 with the specific growth rate of 0.058 d^− 1. Meanwhile, with sucrose, the overall biomass productivity achieved (0.85 g DW L^− 1 d^− 1 ) was 1.18-fold higher than that with glucose. Moreover, a 1.26-fold less doubling time of 9.4 days was noted with sucrose, compared to that in glucose (11.9 days).

3.2 Effect of plant basal medium on biomass production in cell suspension culture of V. odorata

The concentration and composition of nutrients in the basal medium can significantly affect cell growth and metabolism. The carbon source (sucrose 3%, w/v) and plant growth regulator (2,4- D, 3 mg L^− 1, w/v) were kept at identical concentrations in all the tested basal salt combinations. In a separate experiment, a total of 12 different basal media were tested, wherein it was observed that the highest biomass concentration (15.9 ± 0.56 g DW L^− 1) and yield (0.32) could be obtained with MS medium (Fig. 1b) followed by ER medium that produced 13.67 ± 0.48 g DW L^− 1. On the other hand, White medium yielded the lowest biomass concentration (8.9 ± 0.34 g DW L^− 1). Hence, based on these results, for subsequent experiments, MS medium was chosen as the most suitable plant basal medium for cultivating V. odorata cells in suspension.

3.3 Effect of carbon source on biomass production in cell suspension culture of V. odorata

Three different carbon sources, namely sucrose, glucose, and fructose were tested to select a suitable carbon source in the chosen basal medium (MS) for maximum biomass production. Growth curves were plotted for all the carbon sources for up to 22 days to estimate specific growth rates, maximum biomass yield, and productivity. As indicated earlier (section 3.1) in the studies with WPM medium, the same trend was observed with the MS medium supplemented with sucrose (3%, w/v) that yielded the maximum biomass (17.1 ± 0.86 g DW L^− 1) with a specific growth rate of 0.144 d^− 1. Residual sucrose was negligible; however, traces of glucose (0.04 ± 0.01 g L^− 1) and fructose (2.4 ± 0.6 g L^− 1) remained in the spent medium after 12 days of cultivation. Interestingly, the medium pH increased up to 6.7 from the initial value of 5.7, unlike ~ 5.2 observed earlier, which could be attributed to uptake of nitrogen source as discussed later (section 4). The conductivity decreased to 925 ± 162 µS, possibly indicating the nutrient consumption with growth (Fig. 2a).

The results demonstrated that MS medium supplemented with sucrose (3%) results in higher biomass productivity (1.43 g DW L^− 1 d^− 1) than MS medium supplemented with glucose (0.79 g DW L^− 1 d^− 1) or fructose (0.73 g DW L^− 1 d ^− 1). A lag phase of 2 to 8 days of cultivation was also observed when glucose (Fig. 2b) or fructose (Fig. 2c) were used as a carbon source, which could have reduced the biomass productivity. The specific growth rate for glucose and fructose was observed as 0.092 d^− 1 and 0.109 d^− 1, respectively. V. odorata cell suspension cultures grown in sucrose had the lowest doubling time of 4.81 days. In the case of glucose and fructose, the doubling time was observed to be 7.5 and 6.3 days, respectively. Hence, sucrose was designated as the carbon source for further experiments, including the statistical optimization of medium composition.

3.4 Effect of inoculum density on biomass production in cell suspension culture of V. odorata

Inoculum density (ID) is an essential factor that alters cell growth and metabolite production in plant cell cultures. This study demonstrated that as the inoculum density was increased from 3 to 7 g DW L^− 1, biomass yield with respect to the substrate (Y_x/s) also increased from 0.29 g g^− 1 to a maximum of 0.36 g g^− 1 at 7 g DW L^− 1 (ID), after which it decreased to 0.27 g g^− 1 at 11 g DW L^− 1. In addition, the ID of 7 g DW L^− 1 had the highest average growth rate (0.11 ± 0.01 d^− 1), while the ID of 11 g DW L^− 1 had the lowest average growth rate (0.05 ± 0.01 d^− 1) in the tested range (3 to 11 g DW L^− 1). Biomass productivity of ~ 1.5 g DW L^− 1d^− 1 was achieved at an ID of ~ 7 g DW L^− 1 (Fig. 3a). This decrease in biomass growth rate and yield beyond the ID of 7 g DW L^− 1 was presumably due to sucrose limitation, as quantified via HPLC (data not shown).

3.5 Effect of ammonium-to-nitrate ratio on biomass production in cell suspension culture of V. odorata

Apart from carbon source, nitrogen is among the other major nutrients required for growth that is made available in two forms (ammonium nitrate and potassium nitrate) in the MS medium composition (Murashige and Skoog 1962). However, it was known from literature that ammonium-to-nitrate ratio can significantly impact plant cells’ growth and subsequently product formation (Prakash and Srivastava 2005). Many studies have reported the effect of the ammonium-to-nitrate ratio on the growth of plant cell cultures. Hence, different ammonium-to-nitrate ratios were tested from 0:60 to 60:0 mM to select a suitable ratio for maximum growth. As shown in Fig. 3b, maximum biomass (15.6 ± 0.1 g DW L^− 1) was obtained at a ratio of 20:40 (mM), while increasing the ammonium concentration further inhibited the biomass production. Using ammonium as the sole nitrogen source resulted in the lowest V. odorata biomass concentration of 3.9 ± 0.68 g DW L^− 1 (Fig. 3b).

3.6 Plackett–Burman design

Statistical optimization techniques, including PBD and RSM, were applied to find the optimum conditions for maximum biomass production during batch cultivation of V. odorata plant cell suspension. The biomass values obtained after 12 days in the experimental runs was subjected to ANOVA analysis by design expert software. Based on the analysis of the response data, the half-normal plot (Supplementary Fig. S1a) indicated that among the most significant factors, sucrose and potassium nitrate positively influenced the production of biomass. At the same time, ammonium chloride inhibited biomass production in the concentration range studied. The biomass produced in each experimental run, with the respective concentrations of individual nutrient sources is presented in Table 1. It was observed that runs 8 and 11 resulted in maximum biomass, where higher concentrations of sucrose (60 g L^− 1) and potassium nitrate (3800 mg L^− 1) were used along with a lower concentration of ammonium chloride (534.5 mg L^− 1). Similarly, runs 3 and 13 resulted in minimum biomass wherein the lower concentration of sucrose (10 g L^− 1) and potassium nitrate (950 mg L^− 1) were used. In the current study, the model F-value of 19.39 suggests that the model is statistically significant. The derived biomass equation in terms of coded factors is given as follows:

Biomass (g DW L^− 1) = + 8.72 + 3.05A-1.6B + 0.84E-0.81F (2)

where, A is sucrose, B is ammonium chloride, E is potassium nitrate, and F is potassium phosphate monobasic.

p-values less than 0.05 indicated that sucrose and ammonium chloride were significant terms in the model. From Eq. 2, among the studied factors, sucrose was the most significant factor (coefficient value: 3.05) favouring biomass production. Ammonium chloride (coefficient value: -1.6) inhibited biomass production at its higher concentrations, and a similar trend was observed in ammonium-to-nitrate ratio experiment as well (Fig. 3b). Potassium nitrate with a coefficient of 0.84 favoured biomass production with its increasing concentration in the range studied. The predicted R² of 0.69 agrees with the adjusted R² of 0.84. Further, in the current study, CCD was used to optimize the significant nutrient concentrations to achieve the desired response (i.e., biomass concentration) maxima.

3.7 Determination of the optimum concentrations of medium components using RSM

The biomass production from each experimental run of the CCD design is listed in Table 2. The biomass production ranged from 5.1 ± 0.01 g DW L^− 1 to 18.5 ± 1.09 g DW L^− 1. A quadratic biomass production model, including the tested factors' interactive effects, was proposed. The model F-value of 19.05 and p-value of < 0.0001 points toward the model being significant, where sucrose (A), ammonium chloride (B), and potassium nitrate (C) were significant model terms. The model’s R² and adjusted R² values were 0.94 and 0.89, respectively, demonstrating a good fit. An R² value of 0.94 indicates that the model would be unable to justify only about 6% of the total variations in the set. The statistical data revealed that this model was reliable.

The final quadratic equation in terms of coded factors is as follows:

Biomass (g DW L^− 1) = 17.46 + 3.36A-1.52B + 2.34C-0.987AB + 0.66AC + 1.09BC-2.04A²-0.87B²-2.36C² (3)

where A is sucrose, B is ammonium chloride, and C is potassium nitrate.

Sucrose (A) has the highest positive coefficient value of 3.36, demonstrating its positive impact on the response (biomass) and ammonium chloride (B) has the negative coefficient value of 1.52 (represented in Eq. 3), demonstrating it had a negative effect on the response in the concentration range studied. Sucrose and ammonium chloride negatively interacted with a coefficient value of 0.987. The other two interactions [Sucrose (A) and potassium nitrate (C); ammonium chloride (B) and potassium nitrate (C)] had positive coefficient values. Carbon sources have a significant role in biomass production, as reported in the literature (Prakash and Srivastava 2005; Srivastava and Srivastava 2012a). The nitrates positively influenced biomass production, and the same had been observed previously in statistical optimization studies in Azadirachta indica (Srivastava and Srivastava 2012a) for maximizing the production of biomass. The contour plot shows the interactive effect of sucrose, ammonium chloride and potassium nitrate on biomass production (Fig. 4a, b, and c).

Maximum biomass production (18.5 ± 1.1 g DW L^− 1) was obtained in the 18th experimental run of the central composite design, which had 60 g L^− 1 of sucrose. Minimum biomass production (5.1 ± 0.24 g DW L^− 1) was obtained in the 20th and 1st experimental runs. Potassium nitrate (302 mg L^− 1) concentration was lower in run 20, and sucrose concentration (6.3 g L^− 1) was lower in run 1, which resulted in the lowest biomass concentration (5.1 ± 0.24 g DW L^− 1). The residual carbon source analysis for the implemented experimental runs showed that runs 8, 12, 15, 18, and 19 have higher residual fructose concentration (ranging from 21.8 ± 0.13 g L^− 1 to 32.9 ± 3.4 g L^− 1). The optimum concentrations of the three tested nutrients (sucrose, ammonium chloride, and potassium nitrate) were obtained from the numerical optimization tool of the Design-Expert software. The biomass objective function was set to maximum, and other parameters were kept in their tested ranges from the experiments (Table 2).

The model predicted a biomass production of 19.26 g DW L^− 1, verified experimentally with the optimized values of medium components. The optimum concentrations of medium components (sucrose: 45.6 g L^− 1, ammonium chloride: 493.8 mg L^− 1 and potassium nitrate: 2129 mg L^− 1) were predicted from the quadratic model. The cells were inoculated in the model-predicted medium composition. Other medium components and culture conditions were kept as in control conditions. The flasks were harvested (in triplicates) at regular intervals of 2 days till 18 days. On the 12th day, 18.68 ± 0.8 g DW L^− 1 of biomass concentration was obtained (Fig. 4d) with complete glucose utilization from the culture medium (Fig. 4d). The pH of the culture medium did not change significantly and remained stable (5.6 to 5.8) during the exponential growth phase. The electrical conductivity of the medium decreased from 3040 ± 90 µS to 180 ± 20 µS on day 16th, which could be due to the utilization of salts present in the medium. The specific growth rate and doubling time were calculated as 0.16 d ^− 1 and 4.35 d, respectively.

3.8 Effect of rpm on biomass production in cell suspension culture of V. odorata

The shaking speed affects biomass production and cell morphology (González-Cabrero et al. 2018). In the current study, the maximum biomass (18.5 ± 1.0 g DW L^− 1) was obtained at 85 rpm (Fig. 5a). At lower (65 rpm) and higher (165 rpm) values of rpm, biomass production was significantly reduced. Cell sedimentation can occur at lower shaking speeds, limiting the cells' nutrient and oxygen availability in the medium.

At higher rpm, the cell viability is significantly reduced (Fig. 5a). Prakash and Srivastava (2007) reported that A. indica cell death took place in suspension at 200 and 250 rpm and higher shaking speed increases the shear rate, which leads to cell death. In other conditions, the biomass was more or less the same. However, cell viability was much lesser than that at 85 rpm (Fig. 5a). From the carbon source utilization data at 165 rpm, it was evident that 24.3 ± 0.76 g L^− 1 of fructose and 5.1 ± 1.8 g L^− 1 of glucose were present in the spent medium (Data not shown).

Plant cells show a tendency to grow in an aggregated fashion. At lower shaking speeds (65 and 85 rpm), V. odorata cell suspension forms compact cell aggregates (Supplementary Fig. S2a and S2b), whereas, more freely suspended cells with elongated cell morphology were observed at higher shaking speeds (> 125 rpm) (Supplementary Fig. S2d, S2e, and S2f). A similar observation was reported by González-Cabrero et al. (2018) in Pinus pinea embryogenic line 2F47, Fiuk and Rybczyński (2008) in Gentiana kurroo cell suspension culture and Li et al. (2022) in Pinus embryogenic cell suspension culture.

3.9 Effect of temperature and pH on biomass production in cell suspension culture of V. odorata

The biomass production from each experimental run of the CCD design is listed in Table 3. The biomass production ranges from 6.8 ± 1.1 g DW L^− 1 to 19.2 ± 0.4 g DW L^− 1. Lower temperature conditions (< 18.5°C) in runs 8 and 9 resulted in lower biomass concentrations of 7.6 ± 0.5 g DW L^− 1 and 6.8 ± 1.1 g DW L^− 1, respectively. In the case of higher temperature conditions (29.4°C), a maximum biomass concentration of 19.2 ± 0.4 g DW L^− 1 was obtained in run 2. The initial higher pH (7.1) reduced the biomass concentration (13.9 ± 0.2 g DW L^− 1) in run 13, whereas the lower initial pH (4.3) favoured the biomass production (19 ± 1.1 g DW L^− 1) at the identical temperature condition (23°C). Design-Expert software was used for the regression and graphical analysis. A quadratic model incorporating the interactive effects of the factors was proposed for biomass production. The significance of the model is indicated by a model F-value of 28.46 and a p-value of < 0.0001. The model’s R² and adjusted R² values are 0.94 and 0.91, respectively.

The model equation is given as:

Biomass (g DW L^− 1) = 18.43 + 4.71A-1.09B + 0.6750AB-2.95A²-1.43B² (4)

where A is temperature, and B is pH.

It can be inferred that temperature (A) has the highest positive coefficient value of 4.71, and pH (B) has a negative coefficient value of 1.09. Both factors positively interact with a coefficient value of 0.675 (shown in Eq. 4). The contour plot shows the interactive effect of temperature and pH on biomass production (Fig. 5b).

The optimum conditions of temperature and pH were obtained from the numerical optimization tool of the Design-Expert software. Constraints for the optimization were kept within the studied range (Table 3), and the biomass objective was kept as a maximum. The model predicted a maximum biomass production of 20.36 g DW L^− 1. It was verified experimentally with the model-predicted optimum temperature (26.6°C) and pH (5.45) values. The biomass concentration obtained in the experimental run was 20.57 ± 0.73 g DW L^− 1, closely aligning with the model predicted value.

3.10 Effect of light intensity on biomass production in cell suspension culture of V. odorata

Light intensity is a vital factor for cell growth and metabolite production. The maximum biomass production (21.4 ± 0.7 g DW L^− 1) was obtained at 200 Lux (Fig. 5c). In the 2200 to 5200 lux range, biomass production did not change significantly. In completely dark conditions, biomass production was significantly reduced (Fig. 5c). Furthermore, 19.5 ± 1.47 g L^− 1 of fructose was left in the spent medium. In the case of 5200 Lux light intensity, 10.5 ± 0.18 g L^− 1 of residual fructose remained in the culture medium. The utilization of carbon sources indicates the uptake and growth of plant cells.

3.11 Effect of plant growth regulators (PGR) on biomass production in cell suspension culture of V. odorata

PGR molecules can have varied (promoting, inhibiting, or modifying) effects on cell growth and are effective even at lower concentrations (Gaba 2004). Thus, the selection and optimization of concentration for key PGRs can significantly impact in vitro biomass production (Table 4). The ANOVA of the linear model based on data fitting demonstrates an F-value of 5.04, indicating that the selected factorial model is significant, and model terms with p-values < 0.05 indicate confidence in their impact predicted. TDZ was the positively influencing PGR with a coefficient value of 0.2484. GA₃ was the most significant PGR (coefficient value: -0.5328), inhibiting biomass production at higher concentrations. Likewise, 2, 4 – D (coefficient value: -0.3147) and picloram (coefficient value: -0.3872) also inhibit biomass production with its increasing concentration in the range studied (shown in the model Eq. 5). However, as GA₃ and picloram are absent in the control medium (composition described in section 2.2), only thidiazuron (TDZ) was chosen to be taken forward for concentration optimization via response surface methodology, which favoured biomass production (coefficient value: 0.2484) in the range studied. R² and adjusted R² were estimated as 0.78 and 0.63, respectively.

The half-normal plot (Supplementary Fig. S1b) shows the effects of PGRs. Based on these results, the CCD was used further to optimize the TDZ and 2, 4 D concentration to maximize the biomass concentration. 2, 4 D was chosen to be optimized in a lower concentration range as it demonstrated a negative effect on biomass production in the range studied (0.1–0.5 mg L^− 1), which was much lower than that used in the control medium (3 mg L^− 1).

Biomass (g DW L^− 1) = 20.63-0.3147A + 0.0172B-0.0966C + 0.1059D-0.0997E-0.3872F-0.0072G-0.2234H-0.1841J + 0.1072K + 0.2484L-0.5328M + 0.0028N-0.2409O (5)

where A is 2,4 - D, B is IAA, C is IBA, E is IPA, F is Picloram, G is NOA, H is AdS, J is BAP, K is Kinetin, L is TDZ, M is GA3, N is PHC, and O is PG.

3.12 Determination of the optimum concentrations of the selected PGR using central composite designs

PGRs have a significant role in culture growth and metabolism. One or more growth regulators, alone or in combination, lead to regulated metabolism and desired growth. A CCD model can effectively study the interaction between these growth regulators. The full factorial design and the responses obtained from each experimental run are presented in Table 5. The maximum biomass was obtained in run number 9 (TDZ: 1 mg L^− 1 and 2, 4 -D: 0.1 mg L^− 1), and the biomass concentration was 22.5 ± 0.9 g DW L^− 1. The lowest concentration (16.7 ± 0.2 g DW L^− 1) was observed at run number 7. The significance of the model is indicated by the model F-value of 8.50. R² and adjusted R² values are 0.84 and 0.74, respectively. The interaction between the selected PGRs (TDZ and 2,4 -D) is shown in Fig. 6a.

Biomass (g DW L^− 1) = 21.62 + 0.7832A + 1.27B-0.1250AB-0.3021A²-1.30 B² (6)

where A is 2,4 – D and B is thidiazuron.

The optimum concentration of the two PGRs (TDZ and 2,4 - D) tested was obtained from the numerical optimization tool of the Design-Expert software. The biomass objective function was set to maximize, and other parameters were kept in their tested range. The model (Eq. 6) predicted an optimum growth regulator concentration of 0.747 mg L^− 1 of TDZ and 0.1 mg L^− 1 of 2,4 – D; the associated biomass concentration is 22.3 g DW L.

TDZ and 2,4-D favour biomass production in the tested concentration range and negatively interact with a coefficient value of 0.12. From the model (Eq. 6), the optimum solution was obtained and validated experimentally. The batch growth kinetics of V. odorata cell suspension was plotted at the optimized growth regulator concentration. On the 12th day, the biomass concentration was 21.68 ± 0.82 g DW L^− 1 (Fig. 6b). However, the biomass production was similar in the control (3 mg L^− 1 2,4-D alone 21.14 ± 0.53 g DW L^− 1) and the optimized PGR combination (TDZ and 2,4-D − 21.68 ± 0.82 g DW L^− 1). The biomass production and substrate utilization profiles of cell suspension culture, supplemented with optimized medium, are shown in Figure 6b. Glucose (1.12 ± 0.24 g L^− 1) and fructose (12.7 ± 0.79 g L^− 1) were present in the spent medium on day 12. The pH varied between 4.6 to 5.0 during the cultivation period. A decrease in the electrical conductivity (270 µS on day 12) represents the uptake of salts by the cells. The specific growth rate and doubling time were calculated as 0.15 d^− 1 and 4.62 d, respectively. The biomass productivity of the suspension culture of V. odorata cells grown on the optimized medium was 1.8 g DW L^− 1 d^− 1.

3.13 Effect of antifoam on biomass production in cell suspension culture of V. odorata

Plant cell suspension culture has complex protein and lipid molecules like glycoproteins, lipid transfer proteins, phospholipids, and oleosins in the culture medium. These protein complexes favor foaming, as the cell suspension is aerated in the bioreactors by air spargers. Excess foaming reduces growth and influences secondary metabolite production. For this, the current study tested silicone-based antifoam agents (from Hi-Media) at different concentrations. Biomass production and cell viability were measured and compared to control conditions (without antifoam). Antifoam (0.012%, v/v) did not reduce biomass production, and no significant reduction in cell viability could be observed as compared to control conditions (Fig. 6c). The biomass concentration was not affected by higher antifoam concentrations up to 0.12% (v/v). However, the cell viability decreased by up to 60%. Hence, a concentration of 0.012% (v/v) was selected for bioreactor cultivation of V. odorata cell suspension cultures.

3.14 Batch bioreactor cultivation of V. odorata plant cells

Stirred tank reactor (STR)

As shown in Fig. 7a, maximum biomass (19.7 g DW L^− 1) production was observed on day 12, which is 7% lesser than the biomass concentration (21.14 ± 0.53 g DW L^− 1) produced in the shake flasks (data not shown). Presumably, the mass transfer limitations due to foaming and stress developed from the moving parts of reactors could be the reasons behind the reduction in biomass production in this reactor. The sucrose in the medium gets converted into glucose and fructose by enzymatic reactions. The cells preferentially utilize the glucose, which was entirely consumed on day 10. On day 12, the residual fructose concentration was 18 g L^− 1, that showed a value of 12.7 g L^− 1 in the shake flasks. The pH was stable in the range of 5.2 to 5.4. The electrical conductivity dropped to 460 µS from 3170 µS, indicating the utilization of salts. As observed from the growth curve, the cells experience a lag phase from day 0 to day 4. The growth rate and doubling time were calculated as 0.18 d^− 1 and 3.85 d, respectively. The volumetric productivity was calculated as 1.64 g DW L^− 1 d^− 1. The stirrer speed was kept constant at 85 rpm for the initial two days, and then increased to 125 rpm to prevent biomass from settling to the bottom of the reactor vessel. The aeration rate was also increased to 1 vvm to maintain DO above > 20%. On day 12, the viability of cells was found to be 48%.

Airlift reactor (ALR)

The maximum biomass of 14.3 g DW L^− 1 was observed on day 12 (Fig. 7b), which was 11% lesser than the biomass concentration (16.02 ± 0.1 g DW L^− 1) produced in the shake flasks (data not shown). The mass transfer limitations due to high cell density and stress developed from the higher airflow rate are the reasons behind the reduction in biomass production in the airlift reactor. The glucose was consumed entirely on day 12. The fructose concentration remained around 20 g L^− 1 till day 10, which further decreased to 15.4 g L^− 1 on day 12. In shake flasks (100 mL flask), around 18.48 g L^− 1 fructose remained unutilized on day 12. The pH remained constant in the range of 5.2 to 5.4. The electrical conductivity gradually decreased from 3260 µS to 1210 µS during growth. The growth rate and doubling time were calculated as 0.25 d^− 1 and 2.77 d, respectively. The volumetric productivity was 1.2 g DW L^− 1 d^− 1. The initial air flow rate was 0.16 vvm and, after two days, increased to 0.3 vvm. The airflow rate was increased periodically to 1.3 vvm on day 12. As high airflow rates caused intense foaming, the antifoam agent (at an optimised value of 0.012% v/v) was added to the culture broth to control foam formation inside the reactor.

Bubble Column Bioreactor (BCB)

The maximum biomass of 14.5 g DW L^− 1 was observed on day 12 (Fig. 7c), which is 11% lesser than the biomass concentration (16.02 ± 0.1 g DW L^− 1) produced in the shake flasks (data not shown). Presumably, the mass transfer limitations due to high cell density are the reasons behind the reduction in the final biomass concentration achieved in the bubble column reactor. The glucose was entirely consumed on day 12, however the residual fructose concentration was still 18.6 g L^− 1 on day 12. The pH remains constant in the range of 5.4 to 5.8. The electrical conductivity gradually decreased during the growth period from 3420 µS to and reached ~ 1237 µS. The growth rate and doubling time were calculated as 0.303 d^− 1 and 2.28 d, respectively. Volumetric productivity was 1.19 g DW L^− 1 d^− 1. The initial air-flow rate was 0.2 vvm, and during the cultivation, the air-flow rate was adjusted to ensure proper mixing in the system. DO rapidly dropped to 36% on day 4, and the air-flow rate was increased to 0.3 vvm to maintain the DO above 20% through the cultivation period.

3.15 Primary and secondary metabolites present in the V. odorata plant biomass extracts

V. odorata plant biomass was analyzed for the total flavonoids and phenolic compounds using the protocols reported earlier (Folin and Ciocalteau 1927; Wu et al. 2006). V. odorata natural plant and the in vitro plant petiole extracts demonstrated comparable amounts of total phenolics (WVOP:3.8 ± 0.2 mg GAE g DW^− 1, IVOP: 2.7 ± 0.1 mg GAE g DW^− 1). The biomass obtained from the stirrer tank reactor had comparable levels of phenolic compounds (STR:8.42 ± 0.1 mg GAE g DW^− 1) in comparison to that noticed in natural and in vitro plant petiole extracts. Similarly, bioreactor-grown cultures show a similar amount of total flavonoids (5.76 ± 0.3 mg QE g DW^− 1) to natural plant petiole biomass extracts (WVOP: 3.1 ± 0.1 mg QE g DW^− 1 (Fig. 8a).

V. odorata plant biomass extracts were also analyzed with GCMS (Supplementary Fig. S3). The list of metabolites in the biomass extracts is presented in supplementary tables S1, S2, and S3. The metabolites obtained from the bioreactor-grown biomass extracts were compared with those obtained from the natural plant biomass extracts. Out of nearly 60–70 compounds identified in the extracts obtained from the bioreactor-grown biomass (section 2.5.1 and 2.5.2), 18 compounds were found to be in common with those obtained from the natural plant biomass. The list includes bioactive compounds such as stigmasterol (anticancer activity),

5-Hydroxymethylfurfural (food additive), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (antioxidant activity, anticancer agent, antifungal activity), n-Hexadecanoic acid (anti-inflammatory activity), linoleic acid ethyl ester (anti-inflammatory activity), and 3-Deoxy-d-mannoic lactone (antibacterial activity, antifungal activity) as reported in the literature (Sharma et al. 2015). Cyclotides in V. odorata have been reported earlier (Ireland et al. 2006; Narayani et al. 2017a). The cell line VOP-4, used in this study, was analysed for cyclotides earlier (Narayani et al. 2017a). The present study compared bioreactor-cultivated biomass extracts with natural plant biomass extract. The biomass was extracted and analysed with the protocol reported by Narayani et al. (2017a), and the monoisotopic mass (CyO2: 3138.4) was compared with the CyBase (Wang et al. 2008). The total ion chromatogram of natural plant extract and bioreactor cultivated biomass extract were represented in supplementary Fig. S4. The cyclotide (CyO2) was confirmed by its monoisotopic mass values and its late eluting property in reverse-phase chromatography (Koehbach et al. 2013; Burman et al. 2014).

3.16 Antimicrobial activity of V. odorata plant biomass extract.

Multidrug-resistant bacteria pose a threat to human health and also demand the discovery of new antibacterial agents. V.odorata plant extracts have already been reported for their antimicrobial potential (Akhbari et al. 2012; Narayani et al. 2018). In the current study, plant extracts produced from in vitro cultures were tested against respiratory tract pathogens. The natural plant extract (D1) demonstrated potential activity against tested pathogens, and the MIC values ranged from 1.56 to 12.5 mg mL^− 1. The in vitro grown plant (D2) and somatic embryo extracts (D3) had comparable activity to the natural plant extracts. The biomass extracts obtained from bioreactors (D4 and D5) were active against MTCC 3826- Haemophilus influenzae (MIC: D4–6.25 mg mL^− 1 and D5–12.5 mg mL^− 1) and MTCC 655- Streptococcus pneumoniae (MIC: D4–12.5 mg mL^− 1 and D5–12.5 mg mL^− 1). The MIC values were compared to Ampicillin (for H. influenzae – 1.25 mg mL^− 1) as reported in Table 6.

Table 6

Antibacterial activity of *V. odorata* biomass extracts.
S. No	Test organism	Minimal inhibitory concentration (mg mL^− 1)
S. No	Test organism	D1	D2	D3	D4	D5	Ampicillin
1	MTCC 442 - Streptococcus pyogenes	1.56	6.25	12.5	100	100	0.03
2	MTCC 3826- Haemophilus influenzae	12.5	6.25	3.12	6.25	12.5	1.25
3	MTCC 655- Streptococcus pneumoniae	3.12	3.12	3.12	12.5	12.5	-

3.17 Antioxidant activity of V. odorata plant biomass extract

V. odorata primary and secondary metabolites have been reported in the literature for their antioxidant properties. This study compared the antioxidant activity of V. odorata plant extract and its in vitro biomass extracts to Vitamin E (IC50–9094 µg mL^− 1). Among the tested samples, as seen from Fig. 8b, the natural plant extract (D1) had the best scavenging activity with an IC50 value of 647 µg mL^− 1. The in vitro plant extracts (D2: IC50–2640 µg mL^− 1) and STR cultivated biomass extract (D4: IC50–2901 µg mL^− 1) demonstrated comparable activity (Fig. 8b). However, somatic embryo and ALR cultivated biomass extracts showed less (D3: 3-fold and D5: 5-fold) scavenging activity compared to Vitamin E.

For sustainable production of bioactive compounds, a proper understanding of the growth kinetics of plant cell suspensions is crucial, especially in terms of designing plant cell-based bioprocesses and subsequently scaling them up for commercial production. Growth kinetic data, in particular, provides practically relevant insights into the duration of various growth phases, thereby aiding in identifying the optimal batch time for a chosen process. Studying growth kinetics is instrumental in optimizing culture conditions to achieve maximum biomass production. In this context, in the present study, V. odorata plant cell suspension culture produced a maximum biomass concentration (10.2 ± 0.8 g DW L^− 1) on the 12th day at a specific growth rate of 0.074 d⁻¹. Spent medium analysis revealed that around 6.6 ± 0.7 g L^− 1 of glucose and 15.9 ± 0.2 g L^− 1 of fructose were present in the medium. Additionally, the electrical conductivity measurement (1926 µS) indicates the presence of unutilized nutrient salts in the medium. These results show promising leads into effective bioprocess for the selected plant, toward enhanced biomass production. Further optimization can lead to an improvement in biomass productivity and yield.

Different basal salt media have different forms of salts in varying concentrations, with different combinations of vitamins and amino acids. Various such studies are prevalent in literature reflecting on the improved biomass production. The effect of seven different basal media on Cyclocarya paliurus biomass production was studied, and the maximum biomass concentration (17.39 g L^− 1) was obtained in WPM (Yin et al. 2013). Also, Corylus avellana cell suspension culture showed similar culture growth (16.5 g L^− 1) in both MS and B5 medium (Gallego et al. 2015). Six different basal salt media were tested for cultivating Juniperus foetidissima, and the Olive medium was found to be optimum (Qarachoboogh et al. 2022). In the current study, V. odorata cell suspension produced maximum biomass concentration (15.9 ± 0.56 g DW L^− 1) in MS basal medium (Fig. 1b). The higher biomass production in MS medium is possibly due to ammonium nitrate (1.65 g L^− 1) and potassium nitrate (1.9 g L^− 1) present in MS media being higher than in WPM medium (ammonium nitrate 400 mg L^− 1 and potassium sulphate 990 mg L^− 1). Also, ammonium nitrate (1.2 g L^− 1) and potassium nitrate (1.9 g L^− 1) were present in the ER medium, which is relatively higher than other tested basal media, resulting in a biomass concentration of 13.67 ± 0.48 g DW L^− 1. On the other hand, the White medium lacks these or other ammonium salts and has the nitrates in low levels (potassium nitrate 80 mg L^− 1 and calcium nitrate 221 mg L^− 1), producing the lowest biomass concentration (8.9 ± 0.34 g DW L^− 1) among the tested basal media. Thus, MS basal media was shortlisted as the best candidate for further bioprocess studies.

The significant effect of carbon sources on cell growth has been reported in the literature for various plant species. Lantana camara L cell suspension cultures were tested with glucose, maltose, and sucrose for maximum biomass production, wherein sucrose favours biomass (117.8 ± 0.2 g L^− 1) and metabolite (total triterpenoid content: 21 ± 0.6 mg L^− 1) production (Srivastava et al. 2011). In the same study, glucose produced a maximum biomass concentration of 92.8 ± 1.8 g L^− 1 with total triterpenoid content of 10.69 mg L^− 1 (Srivastava et al. 2011). In another report, five carbon sources (sucrose, dextrose, lactose, maltose, and fructose) were tested for Ophiorrhiza mungos, and sucrose favored biomass and metabolite (camptothecin) production at two different concentrations (3 and 5%, w/v). A higher sucrose concentration (5%, w/v) favored camptothecin production (~ 0.22 mg g DW^− 1), whereas lower sucrose (3%, w/v) enabled maximum biomass (Deepthi and Satheeshkumar 2017). Sucrose (3%, w/v) gave the maximum biomass concentration (17.1 ± 0.86 g DW L^− 1) in the current study (Fig. 2a). Glucose and fructose produced lesser V. odorata plant biomass concentration (Fig. 2b and 2c) compared to sucrose. The results observed by Deepthi and Satheeshkumar (2017) in cell suspension cultures of Ophiorrhiza mungos was aligned with the current study, wherein sucrose favoured biomass production, while other monosaccharides led to reduced biomass.

It is well established that higher and lower inoculum densities (IDs) hamper biomass production. A suitable ID can minimize the lag phase and maximize growth rates during batch cultivation (Vasilev et al. 2014). The optimal ID for V. odorata plant cell suspension culture was 7 g DW L^− 1, leading to the highest yield of 0.36 g g^− 1 compared to other tested IDs. Higher and lower inoculum densities reduced V.odorata plant biomass yield upon sucrose (Fig. 3a). Akalezi et al. (1999) studied the effect of ID (1.5 to 6 g DW L^− 1) with a range of sucrose concentrations (30 to 80 g L^− 1). The maximum yield of 0.83 g g^− 1 was obtained with an ID of 3 g DW L^− 1 and 30 g L^− 1 sucrose. Biomass and metabolite production were reported to be affected by ID in Panax notoginseng (Zhang and Zhong 1997) and strawberry cultures (Sakurai et al. 1996). Zhang and Zhong (1997) examined such effects of ID on biomass and total saponin production. The biomass concentration increased as the ID increased from 2.53 to 10.2 g DW L^− 1. However, the yield per unit of sucrose remained consistent at 0.36 g g^− 1. The optimal inoculum size for Panax notoginseng suspension culture was 4.22 g DW L^− 1.

Sakurai et al. (1996) reported that an optimal inoculum of 1 g DW L^− 1 was identified for strawberry cell suspension cultures. The ID for Bacopa monnieri cell suspension culture was optimized using statistical optimization tools, and it was reported that 2 g L^− 1 is a suitable ID (Leonard et al. 2018). The suspension culture of Arnebia benthamii cells with lower ID (5%, w/v) resulted in a higher growth index (~ 5) than with higher ID (15%, w/v) (Kumar et al. 2023). High-density batch cultivations typically require a higher ID as demonstrated in Panax notoginseng cell suspension culture (Zhang and Zhong 1997).

The effect of nitrogen sources, ammonium nitrate and potassium nitrate, was studied in the cell suspension culture of D. carota for anthocyanin production. Ammonium nitrate to potassium nitrate ratio of 20.0:37.6 mM favored biomass (~ 12 g L^− 1 on day 15) and anthocyanin production (2379.99 ± 205.75 mg L^− 1 on day 6). Whereas, only a single nitrogen source in the form of either ammonium nitrate or potassium nitrate hindered the biomass growth (Saad et al. 2018). Similar observations were made in the current study (Fig. 3b), wherein nitrogen from ammonium nitrate significantly reduced the biomass concentration (3.9 ± 0.68 g DW L^− 1). An ammonium-to-nitrate ratio of 20:40 (mM) was suitable for V. odorata plant cell suspension culture, and it produced a biomass concentration of 15.6 ± 0.1 g DW L^− 1 during a 12-day batch cultivation period. In another study, Nothapodytes nimmoniana cell suspension was supplemented with different ammonium-to-nitrate ratios. Nitrate supports biomass formation, while ammonium ions help the metabolite (camptothecin) production. The ratio of 50:10 was found to be optimum for N. nimmoniana cell suspension culture (Karwasara and Dixit 2013). Similar studies were reported for Ophiorrhiza mungos L (Deepthi and Satheeshkumar 2017), Tobacco BY-2 cells (Vasilev et al. 2013), Gymnema sylvestre (Praveen et al. 2011), and Withania somnifera (L.) (Nagella and Murthy 2011).

Plackett–Burman design was used in literature to screen the significant variables that improved the biomass and metabolite production in various plant cell suspension cultures, including Carthamus tinctorius (Liu et al. 2023), Siraitia grosvenorii (Liu et al. 2022), B. monnieri (Leonard et al. 2018) Spilanthes paniculata (Rajendran and Chaturvedi 2017) and A. indica (Prakash and Srivastava 2005; Srivastava and Srivastava 2012a). In the current study, it was observed from Eq. 2 that sucrose and potassium nitrate positively influenced the production of V. odorata plant biomass. Ammonium chloride and potassium phosphate monobasic negatively influenced the biomass production in the tested concentration range (Table 1). Similar observations have been already recorded in the literature pertaining to nutrient sources. For example, Srivastava and Srivastava (2012a) screened the significant nutrient favoring azadirachtin production in A. indica. It was found that sucrose and potassium nitrate positively affected biomass and azadirachtin production with coefficients of 8.69 and 2.37, respectively. Other factors, such as calcium chloride dihydrate and magnesium sulfate hexahydrate, did not affect biomass production. Bansal et al. (2017) reported the same, and their study showed that glucose and potassium nitrate favored biomass production. The selected significant variables (sucrose, potassium nitrate, and ammonium chloride) were optimized with the RSM using Central Composite Design (CCD) or Box–Behnken design protocols.

CCD accounts for the interactive effects (Fig. 4) between the nutrients and their effect on biomass and product production. Optimal solutions were attained with a balance between the product and biomass formation. Also, process economics was improved by adding the optimal concentration of nutrients for plant cell growth and metabolite production. From Eq. 3, the numerical optimization provided the significant nutrient concentrations (sucrose: 45.6 g L^− 1, ammonium chloride: 493.8 mg L^− 1 and potassium nitrate: 2129 mg L^− 1) for maximum biomass production in V. odorata. Sucrose and potassium nitrate concentrations in the optimized medium were higher than in the unoptimized medium. Leonard et al. (2018) used CCD to optimize inoculum size, sucrose, and monopotassium phosphate concentration for maximum Bacopa monnieri biomass and bacoside - A production. The optimized conditions (inoculum size: 2 g L^− 1, sucrose: 30 g L^− 1 and monopotassium phosphate:1.24 mM) produced a maximum biomass of 3.45 g L^− 1 with bacoside A content of 0.49 mg g^− 1.

In another study, the medium composition of A. indica hairy root cultures was optimized using CCD (Srivastava and Srivastava 2012b). The optimization resulted in a biomass concentration of 14.2 g L^− 1 with azadirachtin accumulation of 5.2 mg g^− 1. The optimized medium contains sucrose (40 g L^− 1), potassium nitrate (3.1 g L^− 1), potassium dihydrogen phosphate (0.2 g L^− 1 and magnesium sulfate (0.41 g L^− 1) along with other nutrients at a control level (MS medium composition). CCD was used to optimize the medium composition for cell suspension cultures of Bacopa monnieri (Bansal et al. 2017), Glycine max (Akitha Devi and Giridhar 2014), and Tobacco BY-2 cells (Vasilev et al. 2013). CCD has been a robust tool for optimizing process parameters with a minimal number of experiments.

The shaking speed during in vitro cultivation of plant cells in suspension can impact growth, metabolite production, and shear force on cells (González-Cabrero et al. 2018). In this study, maximum V. odorata plant biomass concentration (18.5 ± 1.0 g DW L^− 1) was obtained at 85 rpm. The literature shows shaking speeds ranging from 70 to 120 rpm for plant cell suspension culture (Motolinía-Alcántara et al. 2021). Picrorhiza Kurroa's cell suspension culture obtained a maximum growth index at 80 rpm (Partap et al. 2022). The effect of shaking speed on Pinus embryogenic cell suspension proliferation was studied, and 70 rpm favored biomass production (Li et al. 2022). Kumar et al. (2023) studied the effect of rpm on an Arnebia benthamii cell suspension culture and reported that a shaking speed of 70 rpm helped the culture growth without any adverse effect on the cell’s physiological state. At higher shaking speeds (> 145 rpm), the viability and biomass concentration of the V. odorata cells was reduced (data not shown). González-Cabrero et al. (2018) observed a decrease in biomass concentration of the cultures growing at 150 rpm rather than at 50 rpm. Presumably, at higher shaking speeds, the hydrodynamic shear stress leads to cell death, and eventually, the biomass concentration is reduced. The optimal shaking speed for a plant cell suspension culture varies based on the plant species and its morphological and physiological properties (Motolinía-Alcántara et al. 2021), thereby demanding species-specific optimization for an effective bioprocess.

Enzymes catalyze most of the biochemical reactions involved in growth and metabolite synthesis. Therefore, optimal temperature and pH conditions are required to achieve maximum rates in these enzymatic reactions, and those optimal conditions are species-specific (Georgiev et al. 2009). Generally, the optimal pH for plant cell culture is established in the range of 5–6 (Grover et al. 2012). The optimal pH for V. odorata cell suspension culture in the current study was 5.45. The effect of pH on Picrorhiza kurroa cell suspension cell growth was studied with the OFAT approach, and it was noted that pH 5.8 favours biomass production (Partap et al. 2022). Withania somnifera cell suspension produced maximum biomass (10.27 g DW L^− 1) at pH 5.8 and withanolide A (2.51 mg g DW^− 1) at pH 6 (Nagella and Murthy 2010). The pH declines during ammonium absorption, and it rises with nitrate uptake by the cells in the medium (McDonald and Jackman 1989). Similar observations were noted in the V. odorata cell suspension culture in the current study. From Eq. 4, temperature and pH had a positive interaction, and the same was presented in Fig. 5b. Numerical optimization with an objective function of biomass maximization resulted in a biomass concentration of 20.36 g DW L^− 1. An increased temperature (26.6°C) favored the biochemical reaction and culture growth. Similar results were obtained and reported on other plant species. The suspension cultures of Nicotiana glutinosa, Populus hybrids, and N. tabacum demonstrated a higher growth rate in the temperature range of 24°C to 30°C (Zhong and Yoshida 1993).

Light is an energy source for photosynthesis, eliciting secondary metabolite production in plant cells (Georgieva et al. 2015). Generally, the optimal light intensity for micropropagation of the plant cell culture has been reported in the range of 60–275 µmol m^− 2 s^− 1 (Batista et al. 2018). It is also reported to have a repressive effect on secondary metabolite (stilbenoid) production (Andi et al. 2019). V. odorata cell and organ cultures have been reported for their cyclotide content (Narayani et al. 2017a, b). The current study obtained the maximum biomass concentration (21.4 ± 0.7 g DW L^− 1) with light intensity of 200 lux. In the literature, it has been reported that the cyclotide content was improved with optimal light intensity. For example, cyclotide Kalata B1 production in Oldenlandia affinis cell culture was improved with optimum light intensity. Lower light intensities (35 µmol m^− 2. S^− 1, approximately equal to 2500 Lux) favored biomass production, and in the case of higher light intensities (120–210 µmol m^− 2. S^− 1), the growth was noticed to be reduced. In another study, Kalata B1 production was similar at 35 and 120 µmol m^− 2. S^− 1 light intensities (Dörnenburg and Seydel 2008).

V. odorata biomass production was unaffected by the different light intensities ranging from 2200 to 5200 Lux (Fig. 5c). Presumably, carbon sources in the culture media favour the plant cells to grow as chemoautotrophs. Similar results were reported by Arias et al. (2016), where the Thevetia peruviana cell suspension cultures used only a carbon source for their growth, and the biomass concentration remained unaffected by the light source.

Hyung et al. (1990) reported a similar observation, where Catharanthus roseus cell suspension culture produced higher catharanthine at lower light intensities (10000 Lux), while photo-inhibition was observed at higher light intensity (20000 Lux). In light and completely dark conditions, Vitis vinifera cell suspension cultures showed no significant differences in biomass concentration, total phenolics and total flavonoids content (Andi et al. 2019), where the cells could utilize the carbon source present in the culture medium for its growth without any demonstrated dependence on the light source.

PGRs have an essential role in plant physiology, including cell division, cell elongation, initiation of flowering, growth of young fruits, fruit ripening, stomatal closure, and development of the abscission zone (Gaba 2004). In plant tissue/cell culture reported for V. odorata, different proportions of PGRs were used to obtain different types of in vitro cultures (Narayani et al. 2017a). The biomass and metabolite production hold prominent scope for improvement using suitable PGR combinations in plant cell suspension (Vasilev et al. 2013). For instance auxins with suitable cytokinins favor the growth of calli and cell suspension.

Numerous PGRs and their combinations were reported in the literature for callus induction (Narayani et al. 2017a) and for the development of cell suspension cultures (Vasilev et al. 2013; Jamwal et al. 2018). As screening designs, PBDs are highly confounded designs that are useful if the interactions (among the factors) are insignificant. In PBD, the main effects are aliased with two-factor interaction. In case of PGRs in plant growth, auxins and cytokinins were known to interact with each other. Hence, a higher resolution IV fractional factorial design was preferred to overcome the PBD’s limitations and to screen the significant PGRs for maximum

V. odorata biomass production. The concentration and ratio of auxin to cytokinin have been essential to maintaining plant cell culture growth. Results from the fractional factorial design of experiments indicated that GA₃ and picloram significantly affected biomass production at the tested concentrations. Subsequently, TDZ and 2,4-D were chosen for further concentration optimization using CCD. The combination of optimized PGRs (TDZ: 0.747 mg L^− 1 and 2,4 – D: 0.1 mg L^− 1) yielded a biomass concentration of 21.68 ± 0.82 g DW L^− 1 during a 12-day batch cultivation period, which was similar to the control conditions (2,4 – D: 3 mg L^− 1). In the control condition, a homogenous and uniform cell suspension was observed. Whereas, with optimized combined PGR conditions, larger cellular aggregates were present on the 12th day. There is no significant improvement in biomass production compared to control conditions. Hence, further experiments used only 2, 4 - D at 3 mg L^− 1.

The effect of PGRs on the cell suspension culture of Tribulus terrestris L was studied, and it was reported that changes in the PGR concentration and ratio significantly affected the culture growth and metabolite production (Klyushin et al. 2022). Replacement of 2,4-D by α-NAA in the culture medium enhances the metabolite diversity (steroidal glycosides), also changing the culture morphology (favors the formation of cellular aggregates) (Klyushin et al. 2022). Similar studies were reported in the cell suspension culture of Melia azedarach L, where the biomass and secondary metabolite production varied with PGR concentration and combinations (Ahmadpoor et al. 2023).

For aeration, the plant cell suspension cultures are sparged with atmospheric air to ensure proper mixing and maintenance of adequate oxygen concentration in the culture medium. During aeration, extracellular protein and polysaccharides cause foaming and hamper mass transfer (oxygen and limited nutrient availability to cells) in bioreactors (Huang and McDonald 2012). The plant cells adhere to the vessel walls and head plate during excessive foaming. Similar observations were made during the cultivation of V. odorata cell suspension culture.

Various antifoam agents were reported in the literature to control the foam in plant cell suspension, while each of those antifoams and their concentration were system-specific. Therefore, a silicone-based antifoam agent (from Hi-Media) was tested at various concentrations (%, v/v) to manage foaming without compromising cell viability. An antifoam concentration of 0.012% (v/v) proved optimal for controlling foam and promoting culture growth. Higher antifoam concentrations (> 0.012%, v/v) decreased the viability and biomass concentration of the V. odorata cell suspension culture. Antifoam C (Dow Corning, US) was used in a similar study to control the foam in cultivating Chenopodium rubrum suspension culture in a photobioreactor (Segečová et al. 2018). Antifoam agents can improve bioreactors' oxygen transfer rate and inhibit culture growth at higher concentrations. N. tabacum bright yellow 2 (BY-2) cells were cultured in STR configuration, and the culture medium was supplemented with pluronic L61 (0.01%, v/v) antifoam agent. The culture grown with pluronic L61 showed lesser growth (40%) and higher metabolite (36%) production in comparison to control conditions (no antifoams).

Interestingly, pluronic L61 compared to its other variants and control (L64, F68 and F127, 211.8 ± 20.7 g L^− 1) showed lesser biomass (127.2 g FW L^− 1) concentration. The study showed that the higher the hydrophobic nature of the antifoam agent, the more is the toxicity toward the BY-2 cells, affecting its cell wall (Opdensteinen and Buyel 2022).

In another study, Reuter et al. (2014) did not use any antifoam agent to cultivate tobacco BY-2 suspension cells in STR configuration (600 L pilot-scale). While the addition of polypropylene glycol to the cell suspension culture of Rubus chamaemorus inhibited its growth (Nohynek et al. 2014), the antifoam SE-15 (0.1%, v/v) added to Cyclopia subternata cell suspension culture prevented the foaming and wall growth in reactor (Kokotkiewicz et al. 2013). These results indicate that the antifoam agent and its concentration must be optimized for each specific plant cell culture.

Stirred tank bioreactor configurations have been preferred for high cell density cultivations, and the shear environment created by the internal moving parts of the reactor enhances the phytochemical production. Established scale-up criteria and the availability of large-scale reactors in industrial setup make the STR a bioreactor configuration preferred for plant cell cultivation. V. odorata cell suspension culture was cultivated in an STR, obtaining a biomass concentration of 19.7 g DW L^− 1 during a 12-day batch cultivation period (Fig. 7a). The STR was equipped with a marine-type impeller to facilitate both axial and radial flow of the culture broth. Furthermore, a stainless-steel sintered sparger aerated the bioreactor with fine air bubbles, enabling improved oxygen transfer.

In another study, Sphaeralcea angustifolia cell suspension culture cultivated in a 2L stirred tank bioreactor equipped with a Rushton-type impeller reached a maximum biomass concentration (19.11 ± 4.34 g L^− 1) on the 11th day of cultivation. Higher (400 rpm) and lower (100 rpm) agitation speeds have been found to reduce biomass production due to hydrodynamic stress and mass transfer limitations (cell settling), respectively. The maximum biomass with suitable metabolite (sphaeralcic acid) production was obtained at 200 rpm (Pérez-Hernández et al. 2019). In a recent report, a stirred tank bioreactor configuration (Pilot scale, 40 L) was used to produce recombinant butyrylcholinesterase (rrBChE) from transgenic rice cell suspension culture. The semicontinuous mode of cultivation (82 days) with suitable K_La values achieved the volumetric productivity of 387 µg L^− 1 d^− 1 (Macharoen et al. 2021). Suitable bioreactor operating conditions, modifications in the agitation system (Srivastava and Srivastava 2012b) and appropriate operating strategies (Srivastava and Srivastava 2012c) are required for specific plant cell cultures for maximum biomass as well as product formation. Motolinía-Alcántara et al. (2021) discussed the vital factors in plant cell bioreactor cultivation and summarized the industrial-scale plant cell bioprocesses.

Airlift reactors(ALRs) require less power when compared to STR and can operate with moderate cell densities. ALR provides good mass transfer with a lower shear environment. Santalum album L and Daucus carota L cell suspension cultures were cultivated in ALR to produce squalene (Rani et al. 2018) and miraculin protein (Park et al. 2020). V. odorata plant cell suspension culture was cultivated in an ALR, incorporating all the optimized conditions directly from shake flask experiments except for the ID. Initial experiments (data not shown) revealed that an ID of 7 g DW L^− 1 led to biomass settling at the bottom of the reactor. Consequently, a reduced ID of 3.5 g DW L^− 1 was implemented for pneumatically driven bioreactors (Fig. 7b). Biomass production was then compared with shake flask cultivation, maintaining an ID of 3.5 g DW L^− 1. Airlift bioreactor configurations could replicate flask-level biomass productivity at a reduced ID.

Seydel et al. (2009) used the medusa-type airlift reactors for Kalata B1 production from Oldenlandia affinis plant cell suspension culture. The maximum biomass concentration was reported to be 9.51 g DW L^− 1. Ageratina pichinchensis cell suspension culture cultivated with 2 L (working volume: 1.7 L) airlift bioreactor and the maximum biomass (11.90 ± 2.48 g L^− 1) was obtained on 11th day (Sánchez-Ramos et al. 2023). In another study, Pueraria candollei var. mirifica cells were cultured in a 5 L airlift bioreactor to produce deoxymiroestrol and isoflavonoid phytoestrogens. Airlift bioreactor cultivated biomass extracts showed a higher metabolite content (deoxymiroestrol: 976 ± 79.6 µg g DW^− 1 and isoflavonoid phytoestrogens: 587 ± 21.6 µg g DW^− 1) than the 2L shake flask cultivated biomass extracts (deoxymiroestrol: 410 ± 53.4 µg g DW^− 1 and isoflavonoid phytoestrogens:159 ± 12.7 µg g DW^− 1) (Udomsin et al. 2020). The difference in the hydrodynamic environment between the shake flasks and the airlift reactor enhances metabolite production (Udomsin et al. 2020). The hydrodynamic environment of different bioreactor configurations (STR, ALR and BCB) was thoroughly studied using Jacaratia Mexicana cell suspension culture (Del Carmen Oliver-Salvador et al. 2013). The results showed that the STR configuration with a higher shear rate influences cysteine protease production. On the contrary, the biomass production (225 g FW L^− 1) was reduced in ALR (2L) cultivation of Arnebia sp suspension culture compared to shake flasks (624.5 g FW L^− 1) (Gupta et al. 2014). The present study observed higher production of secondary metabolites (phenolics and flavonoids) in the STR compared to the ALR (Fig. 8a). ALR configurations were employed in large-scale commercial applications. For example, a pneumatically driven bioreactor system (ProCellExTM system - single-use bioreactor bags designed to provide proper aeration and mixing) was used at Protalix Biotherapeutics to produce recombinant human β-glucocerebrosidase using Daucus carota cell suspension cultures. N. tabacum cell suspension cultures were cultivated with the ProCellExTM system to produce taliglucerase alfa (the first plant-made pharmaceutical approved by the U.S. Food and Drug Administration) (Tekoah et al. 2015).

V. odorata cell suspension culture was cultivated in a BCB and achieved a biomass concentration of 14.5 g DW L. The BCB replicated flask-level biomass productivity at a reduced inoculum density (3.5 g DW L^− 1). Excessive foaming occurred due to the increased aeration rate, which was necessary for proper mixing and maintaining dissolved oxygen concentration (> 20%). In all bioreactor cultivations, cell adhesion on the inner walls was observed because of foaming. This adhesion led to a decrease in biomass concentration and productivity. Pneumatically driven bioreactors can be suitable for cultivating plant cell suspension at a moderate cell density for an extended cultivation period, such as continuous mode cultivation.

Titova et al. (2021) cultivated Dioscorea deltoidei cell suspension culture in a lab scale (20 L) and an industrial scale (630 L) BCB to produce functional food (rich in steroidal glycosides) based on in vitro cultivated biomass. Here, the biomass concentration and cell viability decreased in comparison to shake flask cultivation. The maximum biomass concentration obtained with bioreactors (630 L) was 8.8 ± 2.3 g L^− 1, with 83.5 ± 4.5% cell viability. Phlojodicarpus sibiricus cell suspension culture was cultivated in the bubble column (21 L) and stirred tank (7.5 L) reactor configurations. The low shear environment in the bubble column bioreactor favored higher biomass (15.8 g L^− 1) growth than both STR configuration (8.9 g L^− 1) and shake flask conditions (10 g L^− 1) (Khandy et al. 2021). Eleutherococcus senticosus embryogenic cell suspension was cultured in BCB to produce mature somatic embryos and plantlets (Yang et al. 2012). Here, oxygen transfer with a low shear environment clearly favored the growth of cell and organ cultures in BCB.

Previously, V. odorata leaves were extracted with methanol and analyzed for total metabolites by Aslam et al. (2020). It was reported that the total flavonoid contents in the methanol extract were 7.5 mg QE g DW^− 1, and the total phenolic was 1.06 mg GAE g DW^− 1. The ethanol extract of V. odorata leaves was analyzed, and alkaloids, terpenoids, and saponins were confirmed (Aslam et al. 2020). Akhbari et al. (2012) prepared a methanolic extract from V. odorata aerial parts. The extract was analyzed using GC-MS, revealing the presence of 25 compounds, including linalool, methyl salicylate, and hexadecanoic acid. Cu et al. (1992) reported differences in phytochemical composition between V. odorata leaves and flowers. V. odorata leaves and flowers were extracted using 1,1,2-trichloro-1,2,2-trifluoroethane and hexane. Analysis with GC-MS and GC-FTIR confirmed the presence of 23 compounds. Specifically, V. odorata leaves contained aliphatic hydrocarbons and related oxides.

A previous study done by Narayani et al. (2018) reported that V. odorata plant extracts were active against Escherichia coli (MTCC 443), Staphylococcus aureus (MTCC 737), and Pseudomonas aeruginosa (MTCC 2297) with MIC values less than 50 mg mL^− 1. Akhbari et al. (2012) studied the V. odorata leaf extract against eleven microorganisms, and extracts were reported active against four organisms (P. aeruginosa, E. coli, S. epidermidis, and P. vulgaris) with MIC values less than 0.5 mg mL^− 1. Aslam et al. (2020) have reported the antioxidant activities of V. odorata leaves and the scavenging activity with IC50 values ranging from 160–518 µg mL^− 1. In another study, Akhbari et al. (2012) estimated the antioxidant activity of this plant with IC50 values ranging from 31.5 to 54.7 µg mL^− 1. In the current study, three microorganisms pertaining to respiratory tract diseases were tested, and all of them exhibited growth inhibition upon treatment with extracts from V. odorata. Besides anti-microbial activity, for anti-oxidant activity, natural antioxidant molecules (antioxidant enzymes), phenolic compounds, and flavonoids are required for the plant extract’s efficient action. Variations in the climatic and geographical conditions could result in variations in plant metabolite profiles, resulting in the variation observed in the antioxidant potential in different studies reported. Hence, an integrated approach encompassing various factors discussed above becomes critical for an effective bioprocess to be established for maximum plant cell biomass (and its bioactive compound) production.

It is alarming that many of the industrially important medicinal plants are near extinction or endangered/unavailable in status due to their unregulated harvesting for commercial reasons. The current study focuses on establishing an in vitro plant biomass production strategy as an alternative to natural biomass for V. odorata. Different bioprocess optimization tools and strategies were designed and used in order to maximize biomass productivity (~ 2-fold enhancement), with enhanced yield. For this, different types of bioreactors were used to cultivate the plant cells, of which the STR cultivated biomass showed similarity with the natural plant biomass in terms of its metabolite analysis. This study opens up routes for model-based fed-batch cultivation strategies in STR for further improving the biomass productivity and yield. In addition, the plant metabolite yields can be improved further using elicitors and precursor addition under optimum in vitro conditions. Thus, an optimized plant cell suspension can be scaled up to industrial-scale bioreactors to meet the demand for commercially significant plants like V. odorata for their medicinal applications.

Acknowledgements

Prof. Smita Srivastava would like to acknowledge the financial support under Corporate Social Responsibility (CSR) by Hyclone Life Sciences Solutions India Private Limited (Cytiva) (Project Number: CR22230114BTHLSS008458) and L&T Technology Services (Project Number: CR21221810BTLNTE008458) for the ongoing research in the plant cell technology lab at Indian Institute of Technology Madras including the work stated above.

Babu R would like to acknowledge the Science & Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, Confederation of Indian Industry (CII) and Himalaya Wellness Company for the Prime Minister’s Fellowship Scheme for Doctoral Research (SERB/PM Fellow/CII-FICCI/Meeting/2019).

Author contributions

BR: Methodology, Investigation, Data curation, Visualization, Writing – Original Draft.

SS: Supervision, Funding acquisition, Resources, Conceptualization, Formal analysis, Validation, Writing – Review and editing. All authors read and approved the final manuscript.

Funding

This work was supported by Hyclone Life Sciences Solutions India Private Limited (Cytiva) (Project Number: CR22230114BTHLSS008458) and L&T Technology Services (Project Number: CR21221810BTLNTE008458) under Corporate Social Responsibility (CSR). Author B.R has reveived fellowship from Science & Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, Confederation of Indian Industry (CII) and Himalaya Wellness Company under Prime Minister’s Fellowship Scheme for Doctoral Research (SERB/PM Fellow/CII-FICCI/Meeting/2019).

Competing Interests

The authors report no conflict of interest.

Data availability

All data supporting the findings of this study are available in the paper and its supplementary information section.

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published 07 Aug, 2024

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A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

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Abstract

Figures

Key Message

1. Introduction

2. Materials and Methods

2.1 Developing cell suspension culture of V. odorata

2.2 Growth kinetics of V. odorata cell suspension culture

2.3. Single and multifactor optimization of media components

2.3.1 Selection of plant basal medium for maximum biomass production in cell suspension culture of V. odorata

2.3.2 Effect of carbon source on biomass production in cell suspension culture of V. odorata

2.3.3 Effect of inoculum density on biomass production in cell suspension culture of V. odorata

2.3.5 Plackett–Burman design (PBD) to select the most significant nutrients

2.3.6 Determination of the optimum concentrations of the selected medium components using Response Surface Methodology (RSM)

2.4 Single and multifactor optimization of cultivation conditions

2.4.1 Effect of rpm on biomass production in cell suspension culture of V. odorata

2.4.2 Effect of temperature and pH on biomass production in cell suspension culture of V. odorata

2.4.3 Effect of light intensity on biomass production in cell suspension culture of V. odorata

2.4.5 Determination of the optimum concentrations of the selected PGR using response surface methodology (RSM)

2.4.6 Effect of antifoam on biomass production in cell suspension culture of V. odorata

2.5 Batch bioreactor cultivation of V. odorata plant cells

2.5.1 Stirred tank reactor (STR)

2.5.2 Airlift bioreactor (ALR)

2.5.3 Bubble column bioreactor (BCB)

2.6 Analytical methods

2.6.1 Cell viability assay

2.6.2 Estimation of residual carbon source using high-performance liquid chromatography (HPLC)

2.6.3 Estimation of total flavonoid and total phenolics

2.6.4 GCMS and LCMS analysis of V. odorata plant biomass

2.7 Bioactivity analysis of V. odorata plant biomass extracts

2.7.1 Antimicrobial activity of V. odorata plant biomass extract

2.7.2 Antioxidant activity of V. odorata plant biomass extract

2.8 Statistical analysis

3. Results

3.1 Growth kinetics of V. odorata cell suspension culture

3.2 Effect of plant basal medium on biomass production in cell suspension culture of V. odorata

3.3 Effect of carbon source on biomass production in cell suspension culture of V. odorata

3.4 Effect of inoculum density on biomass production in cell suspension culture of V. odorata

3.5 Effect of ammonium-to-nitrate ratio on biomass production in cell suspension culture of V. odorata

3.6 Plackett–Burman design

3.7 Determination of the optimum concentrations of medium components using RSM

3.8 Effect of rpm on biomass production in cell suspension culture of V. odorata

3.9 Effect of temperature and pH on biomass production in cell suspension culture of V. odorata

3.10 Effect of light intensity on biomass production in cell suspension culture of V. odorata

3.12 Determination of the optimum concentrations of the selected PGR using central composite designs

3.13 Effect of antifoam on biomass production in cell suspension culture of V. odorata

3.14 Batch bioreactor cultivation of V. odorata plant cells

3.15 Primary and secondary metabolites present in the V. odorata plant biomass extracts

3.16 Antimicrobial activity of V. odorata plant biomass extract.

3.17 Antioxidant activity of V. odorata plant biomass extract

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1