Optimization of spinosad production by Saccharopolysosa spinosa YJY-12 and its application in controlling Eggplant Thrips

Abstract

In this study, a new isolated strain YJY-12 with high yield of spinosad was isolated and its fermentation conditions for spinosad production was optimized. It was preliminary identified by morphological and molecular biology analysis to be S. spinosa YJY-12, and it demonstrated stable high-yield trait, with a variation of less than 5% in fermentation levels across five generations. Subsequently, its fermentation condition for producing spinosad was optimized in shake flask experiments. The process involved single-factor tests, Plackett–Burman (PB) experiments, steepest ascent experiments, and central composite design (CCD) methodology. After optimization, the spinosad fermentation level by strain YJY-12 reached up to 4.38 g/L. The fermentation process was further scaled up to a 30-L fermentor by fed-batch fermentation, achieving a spinosad fermentation level of 6.22 ± 0.12 g/L after 16-day. Finally, the efficacy of spinosad in controlling Eggplant Thrips was investigated and the technical spinosad produced by strain YJY-12 exhibited superior efficacy of thrips control. Overall, this study provided a methodological foundation for enhancing spinosad fermentation efficiency and offers guidance for future spinosad fermentation optimization research.

Introduction

Spinosad, a macrolide-based biological insecticide produced by the aerobic fermentation of Saccharopolyspora spinosa¹, was developed in the 1990 s by Dow AgroSciences to tackle agricultural pest control challenges. It consists primarily of spinosyn A (85–90%) and spinosyn D (10–15%)². Renowned for its efficacy and environmental compatibility, spinosad was awarded the US Presidential Green Chemistry Challenge Award in 1999³. Its insecticidal mechanism, which does not exhibit cross-resistance to other pesticides⁴, involves binding to nicotinic acetylcholine receptors (nAChRs), causing nerve cell depolarization, hyperactivation of the central nervous system, muscle failure, and death⁵. Additionally, it also inhibits γ-aminobutyric acid (GABA) receptor activation, further enhancing its insecticidal activity⁶. As a broad-spectrum insecticide, it effectively controls Lepidoptera, Diptera, and Thysanoptera pests, and shows toxicity to specific Coleoptera, Orthoptera, Hymenoptera, Isoptera, and Nematodes species^7–9.

Although spinosad holds great application potential, its industrial production encounters challenges mainly from low-productivity bacterial strains and an immature fermentation process¹⁰. Therefore, improving spinosad yield has become critical, with enhancing fermentation performance of strain by breeding techniques being a key strategy. Physical and chemical mutagenesis like UV irradiation^11–13 and ARTP/NTG¹⁴ exposure are common for breeding high-yielding strains. Other breeding techniques such as metabolic engineering¹⁵, introduction of regulatory genes¹¹, genome-scale metabolic model guided engineering¹⁶, genome shuffling¹⁷, and the heterologous biosynthesis¹⁸ have also been employed. Meanwhile, optimizing fermentation processes is vital method to improve the level of spinosad fermentation. Strobel and Nakatsukasa¹⁹ used response surface methodology (RSM) for medium optimization, raising macrolide production to 1 g/L, a 25-fold increase. Research showed optimal glucose (58.8 g/L) and phosphate concentrations (29.41 mM) could lead to 507 mg/L spinosad production in 5 L fermentation²⁰. Models were developed for predicting biomass and substrate levels have effectively captured the spinosad fermentation process¹². All the aforementioned studies utilized glucose as the main carbon source. Yang et al.²¹, optimized the spinosad production medium by using RSM method, developing an optimal medium with mannitol as the main carbon source. As spinosad is an aerobic secondary metabolite, oxygen deficiency during fermentation can constrain its production. To address this, Luo²² et al., found that expressing the Vitreoscilla hemoglobin gene could enhance oxygen uptake and spinosad yield. Moreover, Bai²³ et al., established a four-stage dissolved oxygen (DO) strategy, which significantly increased spinosad production relative to constant DO control.

In this study, a novel spinosad-producing strain YJY-12 was initially identified by morphological and molecular biological analyses. The inheritance and fermentation stability of S. spinosa YJY-12 were then examined. Furthermore, single-factor experiments, PB experiments, steepest ascent experiments, and response surface methodology experiments were conducted to systematically investigate the cultivation conditions and optimal medium composition for spinosad production. A 30 L fermenter was employed to produce spinosad by fed-batch fermentation. Finally, the effectiveness of spinosad derived from S. spinosa YJY-12 in controlling Eggplant Thrips was assessed.

Results and discussion

Morphological and molecular biology analysis of strain YJY-12

From soil samples, we isolated 542 actinomycetes based on colony morphology and color (The process for strain isolation was detailed in the supplementary materials). HPLC analysis revealed that only 76 of these strains produced spinosad, with nearly five-sixths exhibiting fermentation levels below 500 mg/L. The top 5 spinosad-producing strains are listed in Table S1. Notably, the newly isolated strain YJY-12 demonstrated a notably high spinosad yield, achieving a fermentation level of 2.45 g/L. As shown in the Fig. 1A, the spinosad A accounted for over 95% of the spinosad A/D proportion and the ratio of spinosad A: spinosad D was up to 21:1. In the insecticidal spectrum of spinosad, the insecticidal activity of spinosad A was higher than spinosad D, indicating that the spinosad, produced by strain YJY-12, showed better insecticidal activity²⁴. A further study of strain YJY-12 was investigated by morphological and molecular biology identification. The strain YJY-12 was incubated into solid culture medium and cultivated at 30 °C for 7 days to form colonies, as shown in Fig. 1B. The strain YJY-12 showed white colony with a diameter of approximately 2–3 mm and neat edges. There were aerial hyphae and spores, but no pigment was produced. After 3 days of liquid culture, the mycelial morphology of strain YJY-12 was shown in Fig. 1C. The mycelium of strain YJY-12 extended and showed agglomerates and radial with branches and septa. The colony and mycelium morphology were accorded with the characteristics of Saccharopolyspora spinosa.

Fig. 1.

The HPLC chromatogram of spinosad produced by strain YJY-12 and the culture characteristics of strain YJY-12 in medium. (A) The HPLC chromatogram of spinosad produced by strain YJY-12; (B) Colony morphology of strain YJY-12 in solid medium; (C) Microscopic morphology of strain YJY-12 in liquid medium.

The 16S rRNA gene sequence of strain YJY-12 was obtained by PCR, submitted to GenBank (accession number: PP998559), and blasted with the NCBI database. Its 16S rRNA gene sequence showed 99.79% identity with strain Saccharopolyspora spinosa NRRL 18395^T (AEYC01000092)²⁵ which belonged to the genera of Saccharopolyspora. Phylogenetic relationship between strain YJY-12 and its high 16S rRNA sequences similarity strains was evaluated by MEGA 11²⁶. It could be observed that strain YJY-12 was situated in the same clade with Saccharopolyspora spinosa NRRL 18395^T (AEYC01000092) from the phylogenetic tree based on 16S rRNA gene sequence (Fig. 2A). Thus, based on morphological and phylogenetic analysis, the strain YJY-12 was preliminarily identified to be a strain of Saccharopolyspora spinosa and named as Saccharopolyspora spinosa YJY-12.

Fig. 2.

Phylogenetic analysis and genetic stability of S. spinosa YJY-12. (A) Phylogenetic tree inferred from Neighbour-Joining analysis of 16S rRNA sequence of YJY-12 and its closest matches in the GenBank. (B) The genetic stability of S. spinosa YJY-12.

Analysis of inheritance and ferment stability of S. spinosa YJY-12

Streptomyces were prone to genetic instability, which may lead to degradation of high-yielding strains during subculture, resulting in lower fermentation levels during fermentation²⁷. The strain S. spinosa YJY-12 was subjected to successive subculturing for a total of five passages. The fermentation capability of spinosad produced by each subsequent generation of S. spinosa YJY-12 was shown in Fig. 2B.

After five generations of continuous transfer, it was found that S. spinosa YJY-12 could stably inherit the trait of high production of spinosad. The fermentation level of strain after passage showed a relatively small change, with a variation of less than 5% in the fermentation level. However, actinomycetes were prone to degeneration, so it was necessary to rejuvenate the strain regularly to ensure its stable fermentation level.

Optimization of fermentation conditions for spinosad production

Screening of initial fermentation medium

In order to screen the initial fermentation medium, nine different kinds of fermentation media were preliminary selected. As shown in the Fig. 3A, formula 1 showed highest spinosad fermentation level with 2.45 g/L, followed by formula 4 with 2.05 g/L. Therefore, formula 1 was selected as the initial fermentation medium for the following medium optimization.

Fig. 3.

Effect of different factors on spinosad production. (A) Effect of different initial fermentation medium on spinosad production; (B) Effect of seed-age on spinosad production; (C) Effect of inoculation volume on spinosad production; (D) Effect of fermentation temperature on spinosad production; (E) Effect of initial medium pH on spinosad production; (F) The effect of culture time on spinosad production.

The effect of seed-age on spinosad production

Different seed-age (24–84 h) were selected and the seed solution was transferred into the initial fermentation medium with inoculation volume 10% culture for 10 d. As shown in the Fig. 3B, the fermentation level of spinosad increased sharply between 24 and 60 h of seed age, reaching maximum at 60 h with a yield of 2.9 ± 0.08 g/L. Continuing to prolong the seed age will have a significant inhibitory effect on the synthesis of spinosad.

The effect of inoculation volume on spinosad production

Generally, the inoculation volume was often ranged from 5 to 20% in antibiotic fermentation. Therefore, the inoculation volume (2.5–15%, v/v) were selected in this experiment, and the fermentation level of spinosad was detected after 10 days. As shown in the Fig. 3C, with the inoculation volume increasing from 2.5 to 10%, the spinosad yield increased sharply. When the inoculation volume was 10%, the spinosad yield reached maximum level with 2.95 ± 0.12 g/L. Afterwards, as the inoculation volume increased, the spinosad yield decreased and the fermentation broth became viscous. During the fermentation process, when the seed age was short and the inoculation volume was small, the amount of mycelium was small, and the mycelium clumped and formed balls. With the increase of inoculation volume and seed age, due to the limitation of dissolved oxygen, the mycelium extended around in a linear way to obtain larger dissolved oxygen and it gradually developed from spherical to filamentous, resulting in the rheology properties of the culture medium changed from Newtonian fluid to non-Newtonian fluid, and the latter, due to the increase in medium viscosity, affected normal ventilation and mass transfer, resulting in a significant decrease in dissolved oxygen²⁸.

The effect of fermentation temperature on spinosad production

The synthesis of antibiotics and bacterial growth were carried out under the catalysis of various enzymes with appropriate temperature. Selecting an appropriate temperature was crucial for maintaining microbial growth and antibiotic synthesis²⁹. In order to optimize the fermentation temperature, the cultivation temperature was set at 26–34 °C. The results were shown in Fig. 3D. The results showed that as the temperature increased from 26 to 30 °C, the fermentation level of spinosad increased gradually and then decreased from 30 to 34 °C. Therefore, the optimal fermentation temperature was 30 °C. The fermentation temperature of spinosad was often set at 28–30 °C^17,22,30. The cultivation temperature was set at too low or too high, which affected the growth of mycelium and subsequently affects the synthesis of spinosad.

The effect of initial medium pH on spinosad production

The pH in the fermentation environment showed significant impact on the growth and metabolism of microorganisms³¹. In this experiment, the initial pH of the fermentation medium was adjusted to different values to investigate its impact on spinosad production. The experimental results are shown in Fig. 3E. As shown in the Fig. 3E, the optimal initial pH for product synthesis was 7.0. The pH of the initial fermentation medium was too low or too high, which is not conducive to the synthesis of spinosad. The pH of the initial fermentation medium was set at 6.5–7.5 which was beneficial for the growth of S. spinosa^12,19. And the pH suitable for antibiotic fermentation was generally between 5.0 and 8.0.

The effect of culture time on spinosad production

For optimization culture time of S. spinosa YJY-12, the strain was incubated in the fermentation medium with seed-age 60 h, inoculation volume 10%, fermentation temperature 30 °C and initial medium pH 7.0. After 5 days of cultivation, the fermentation level of spinosad was detected and then the fermentation level was detected once a day. The results were shown in the following Fig. 3F. As shown in Fig. 3F, in the early stage of fermentation (5–11 days), with the extension of fermentation time, the fermentation level of spinosad rapidly increased. This was because the culture medium at this stage was rich in nutrients, and the strain showed great metabolic activity. At 11-days of fermentation, the highest the fermentation level of spinosad reached 3.04 ± 0.06 g/L. Then, the fermentation level of spinosad began to decrease gradually. It may be due to the depletion of nutrients in the culture medium, which affected the production of spinosad.

Plackett–Burman experiment

Software Design expert 8.0.6 was used to analyze the results of the Plackett–Burman (PB) experiment, which are presented in Table 1. The best-fit equation obtained is as follows:

where FA indicated the fermentation level of spinosad, A, B, C, D, E, F, G, H and I represented glucose, glycerol, methyl oleate, soybean oil, casein peptone, yeast extract, cottonseed meal, corn gluten meal, and corn steep liquor dry powder respectively.

Table 1.

The results of PB experiment.

Number	A	B	C	D	E	F	G	H	I	Fermentation level of spinosad (g/L)
1	− 1	− 1	1	− 1	1	1	− 1	1	1	2.54
2	− 1	− 1	− 1	1	− 1	1	1	− 1	1	1.48
3	1	− 1	1	1	− 1	1	1	1	− 1	3.72
4	− 1	1	− 1	1	1	− 1	1	1	1	2.4
5	1	− 1	− 1	− 1	1	− 1	1	1	− 1	3.28
6	1	1	− 1	− 1	− 1	1	− 1	1	1	2.4
7	− 1	1	1	1	− 1	− 1	− 1	1	− 1	1.82
8	1	1	− 1	1	1	1	− 1	− 1	− 1	2.84
9	− 1	− 1	− 1	− 1	− 1	− 1	− 1	− 1	− 1	1.03
10	− 1	1	1	− 1	1	1	1	− 1	− 1	3.4
11	1	1	1	− 1	− 1	− 1	1	− 1	1	3.89
12	1	− 1	1	1	1	− 1	− 1	− 1	1	3.58

The model was evaluated using Fisher’s test and showed a high probability P-value of 0.0263. The coefficient of determination (R²) was 0.9940, and the adjusted R² (adj R²) was 0.9669, indicating the model’s validity. The highest spinosad fermentation level of 3.89 g/L was observed in medium 11.

The PB experiment results (Table 2) indicated the following order of factor significance for spinosad production: A > C > G > F > E > B > D > I > H. Factors with significant effects (p < 0.05) were glucose, methyl oleate, cottonseed meal, and casein peptone, while others showed no significant impact. Consequently, these four factors were chosen for further optimization. The experimental data revealed that the fermentation level of spinosad increased with higher concentrations of these four factors, demonstrating a positive correlation between their concentrations and spinosad production.

Table 2.

The analysis results of the PB experiment.

Factors	t	Prob > F	Order of importance
A (Glucose)	+ 0.59	0.0067	1
B (Glycerol)	+ 0.093	0.1935	5
C (Methyl oleate)	+ 0.46	0.0109	2
D (Soybean oil)	− 0.058	0.3512	6
E (Casein peptone)	+ 0.31	0.0237	4
F (Yeast extract)	+ 0.032	0.5800	7
G (Cottonseed meal)	+ 0.33	0.0208	3
H (Protein powder)	− 0.005	0.9271	9
I (Corn steep liquor powder)	+ 0.017	0.7633	8

Steepest ascent experiment

The steepest ascent experiment results were shown in Table 3. The spinosad production of experimental groups showed an increasing trend with the increase concentration of glucose, methyl oleate, cottonseed meal and casein peptone. Medium 6 showed highest fermentation level of spinosad with 3.60 g/L, whereas spinosad production of medium 7 decreased slightly. Thus, the medium 6 was chosen for optimization through the response surface method.

Table 3.

The results of steepest ascent experiment.

Group	Glucose (g/L)	Methyl oleate (g/L)	Cottonseed meal (g/L)	Casein peptone (g/L)	Fermentation level of spinosad (g/L)
1	40	32	20	7.5	2.52
2	44	35	24	9	2.73
3	48	38	28	10.5	3.05
4	52	41	32	12	3.14
5	56	44	36	13.5	4.10
6	60	47	40	15	3.90
7	64	50	44	16.5	3.54

Central composite design method

The fermentation medium was optimized using a central composite design, and the results are presented in Table 4. Software Design expert 8.0.6 was used to analyze the data, and the quadratic polynomial regression equation was obtained as follows:

Table 4.

The design and results of response surface method.

Experimental run	A (Glucose)	B (Methyl oleate)	C (Cottonseed meal)	Fermentation level of spinosad (g/L)
1	− 1	0	− 1	1.78
2	0	1	− 1	3.22
3	− 1	1	0	2.13
4	0	− 1	1	3.17
5	1	1	0	3.74
6	− 1	− 1	0	1.54
7	1	0	− 1	2.31
8	0	1	1	2.85
9	1	− 1	0	2.21
10	0	0	0	4.15
11	0	0	0	4.25
12	1	0	1	4.28
13	0	− 1	− 1	2.16
14	− 1	0	1	2.45
15	0	0	0	4.21
16	0	0	0	4.16
17	0	0	0	4.18

In this model, the predicted fermentation level of spinosad was denoted as FA, while A, B, and C represent the concentrations of glucose, methyl oleate, and cottonseed meal, respectively. The ANOVA results for the model were presented in Table 5. The model was found to be highly significant (p = 0.0008), with R² = 0.9524 and Adj-R² = 0.8913, indicating a good fit to the experimental data and good predictive capabilities. The linear terms for glucose (A), methyl oleate (B) and cottonseed meal (C), as well as the square terms for glucose (A²), methyl oleate (B²), and cottonseed meal (C²), were significant (p < 0.05). This implies that these variables have a substantial impact on spinosad production. The interactions between factors were not significant, as shown by the p-values for AB (0.1930), AC (0.0867), and BC (0.0723). Accordingly, it could be inferred that the main affecting spinosad production were in the order of A > C > B.

Table 5.

ANOVA for response surface quadratic model.

Source	Sum of squares	df	Mean square	F value	p value	Significancy
Model	14.92	9	1.62	15.57	0.0008	**
A-Glucose	2.69	1	2.69	25.27	0.0015	**
B-Methyl oleate	1.02	1	1.02	9.60	0.0174	*
C-Cottonseed meal	1.34	1	1.34	12.63	0.0093	**
AB	0.22	1	0.22	2.07	0.1930
AC	0.42	1	0.42	3.97	0.0867
BC	0.48	1	0.48	4.47	0.0723
A2	3.92	1	3.92	36.82	0.0005	**
B2	2.83	1	2.83	26.59	0.0013	**
C2	1.14	1	1.14	10.69	0.0137	*
Residual	0.75	7	0.11
Lack of Fit	0.74	3	0.25	149.26	0.0001	**
Pure Error	6.600E − 0.03	4	1.650E − 0.03
Cor Total	15.67	16

*p < 0.05; **p < 0.01.

The response surface plot of spinosad production showed the interactive effects of glucose, methyl oleate, and cottonseed meal in spinosad production (Fig. 4). As shown in Fig. 4, the fermentation level of spinosad tended to increase first and then decrease with the variation of each factor. The optimal fermentation level was located at the top of each plane, and the interaction of the factors was not significant. At the stationary point, a theoretical maximum value of spinosad production was 4.33 g/L and the concentration of glucose, methyl oleate and cottonseed meal was 56.52 g/L, 44.39 g/L and 36.52 g/L respectively. Considering the experimental feasibility, the concentration of glucose, methyl oleate and cottonseed mealwe adjusted the optimal degradation conditions were adjusted to 56 g/L, 44 g/L and 37 g/L. The three repeated experiments were carried out under the optimal combination. The results demonstrated that fermentation level of spinosad reached 4.35 g/L, 4.28 g/L, and 4.42 g/L. The average value was 4.38 g/L, which was very close to the theoretical value.

Fig. 4.

The effect of glucose (A), methyl oleate (B) and cottonseed meal (C) on spinosad production (E).

Fed-Batch fermentation

Fed-batch fermentation was the most effective way to improve the yield of target product. Fed-batch fermentation could significantly extend the stationary phase of fermentation, which was conducive to the accumulation of secondary metabolite. However, the supplementation of carbon sources in antibiotic fermentation was a double-edged sword. The lack of carbon sources in the fermentation medium led to hyphal rupture and entered decline period in advance, directly affecting the synthesis of products. Excessive sugar supplementation caused secondary growth of hyphae and the metabolic flow allocation focused on primary metabolism, producing substrate inhibitory effects which were not conducive to the production of secondary metabolites^32–34. The Fed-batch culture curve of 30 L fermentation tank was shown in Fig. 5.

Fig. 5.

Fed-Batch fermentation of spinosad production by 30 L fermentation tank.

As shown in Fig. 5, the pH of the fermentation broth increased, indicating that strain YJY-12 consumed amino acids in the medium for growth, thereby releasing ammonium ions. During the early fermentation phase (0–4 days), the increase in spinosad fermentation levels was relatively slow. This was attributed to the rapid mycelial growth and the comparatively slower progression of secondary metabolism during the logarithmic growth phase. Upon reaching the stationary phase (4–16 days), the wet mycelium attained 50% by day 4, followed by a rapid increase in spinosad fermentation levels. On day 5, the reducing sugar concentration was measured at 24 g/L, which prompted the initiation of glucose supplementation. By adding 80% (v/v) glucose, the reducing sugar concentration in the fermentation medium was effectively maintained within the range of 10–15 g/L. The fed-batch conditions were found to be appropriately set. Under the aforementioned fermentation conditions, the fermentation performance demonstrated a steady upward trend, and the control of fermentation conditions was relatively uncomplicated. Additionally, S. spinosa YJY-12 did not exhibit secondary growth, resulting in a prolonged stationary phase of up to 9 days. After 16 days of fermentation, the spinosad fermentation level reached 6.22 g/L, representing a 42% increase compared to that achieved through shaking flask fermentation. Therefore, the spinosad, produced by the aerobic fermentation of S. spinosa, was particularly suitable for fed-batch fermentation.

Many studies have been dedicated to enhancing spinosad production by optimization of fermentation processes, with a important factor on selecting medium components for optimal growth and spinosad yield of Saccharopolyspora spinosa. Strobel and Nakatsukasa¹⁹ developed a response surface methodology to optimize the composition of fermentation medium and to characterize the microorganism’s response to systematic variations in medium composition. The overall increase in fermentation level of spinosad from the original fermentation medium to the optimized medium was over 25-fold. Liang¹² et al., reported a high spinosad production strain UV-42-13 and created the model of the fermentation process in order to predict biomass, substrate, and fermentation level of spinosad, during the fermentation process. The highest level of spinosad fermentation by this strain has been found to reach 177.8 mg/L and the developed model could well express the fermentation process of spinosad. Moreover, Yang²¹ et al. used response surface method to optimize the fermentation medium and the optimized fermentation medium was used to screen high yielding strain of spinosad. The highest spinosad yield strain showed the fermentation level of spinosad from 547 mg/L to 1035 ± 34 mg/L. Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. A four-phase DO strategy for spinosad fermentation was established. DO was maintained at 40% during 0–24 h, 50% during 24–96 h, 30% during 96–192 h, and 25% until the end of fermentation²³. The four-stage DO strategy resulted in an increase in spinosad levels of 652%, 326%, 546%, and 781% compared with constant DO control at 50%, 40%, 30%, and 20%, respectively.

Extraction of spinosad from fermentation broth

Four liters of fermentation broth underwent a series of purification steps, including solid–liquid separation, methanol extraction, concentration, sec-butyl acetate extraction, tartaric acid back-extraction, alkali precipitation, methanol redissolution, and recrystallization, ultimately yielding technical spinosad. The spinosad content and recovery rate at each extraction step were presented in Table 6. Notably, in the spinosad extraction process, the recovery yield at each step, excluding crystallization, exceeded 95%. The overall recovery yield achieved was 89%. The final product showed a purity of 91%, with the ratio of spinosad A/D reaching 30: 1 (Fig. S1).

Table 6.

Extraction of spinosad from fermentation broth.

Extraction steps	Spinosad content (g)	Recovery rate (%)
Fermentation broth	26.35
Methanol extraction	25.91	98.32
Sec-butyl acetate extraction	25.87	99.86
Tartaric acid back-extraction	25.08	96.94
Alkali precipitation	25.08	100
Recrystallization	23.48	93.62

Experimental study on the efficacy of spinosad in controlling Eggplant Thrips

The efficacy of spinosad in controlling Eggplant Thrips was shown in Table 7. Notably, the spinosad produced by S. spinosa YJY-12 showed superior rapid control at lower doses. After a single day of spraying pesticides with a 10 g/15 L dose, it achieved 77.51% efficacy, outperforming the 57.01% efficacy of spinosad from QILU. At a higher dose of 15 g/15 L, the efficacy of YJY-12’s spinosad rose to 87.93%, closely matching the 87.75% efficacy of the commercial spinosad preparation, while QILU’s product reached only 78.12%. Three days post-application, the spinosad produced by S. spinosa YJY-12 maintained 75.90% efficacy at the 10 g/15 L dose, and 84.62% efficacy at the 15 g/15 L dose, compared to 60.73% and 65.69% for QILU’s product, respectively. The commercial preparation showed 77.65% efficacy at the higher dose. After 7-days, YJY-12’s spinosad maintained 62.65% efficacy at the lower dose and 83.34% at the higher dose, while QILU’s efficacy was 62.11% and 67.25%, respectively. The commercial preparation’s efficacy was 72.66% at the higher dose. Consequently, it was evident that the technical spinosad produced by S. spinosa YJY-12 demonstrated better long-term efficacy of thrips control.

Table 7.

Field efficacy trial results of Thrips insecticide.

Spinosad preparation	Dosage (g/L)	Efficacy of thrips control/%
		1 day after spraying	3 days after spraying	7 days after spraying
Spinosad purchased from QILU	10	57.01	60.73	62.11
	15	78.12	65.68	67.25
Spinosad produced by S. spinosa YJY-12	10	77.51	75.90	62.65
	15	87.93	84.62	83.34
Commercial spinosad preparation	15	87.75	77.65	72.66

In conclusion, the spinosad from S. spinosa YJY-12 exhibited the highest efficacy in controlling Eggplant Thrips, surpassing both QILU’s technical spinosad and the commercial preparation, particularly in the long term.

Summary

In this study, Saccharopolyspora spinosa YJY-12, a high-yielding spinosad-producing strain, was identified by morphological and molecular biological analyses. This strain exhibited a high fermentation level of 2.45 g/L of spinosad, with an A/D ratio of 21. Moreover, it displayed stable high-yielding traits over five generations, with a fermentation variation of less than 5%. The optimized fermentation condition was as follows: seed-age 60 h, inoculation volume 10%, 30 °C, pH 7.0, and culture time 11-days. Furthermore, the fermentation medium was optimized using PB tests, steepest ascent experiments, and CCD. Under those optimized conditions, the spinosad fermentation level of strain YJY-12 reached 4.38 g/L after 11-days. By employing fed-batch fermentation, the spinosad fermentation level peaked at 6.22 ± 0.12 g/L after 16-days in 30-L fermenter. Notably, the technical spinosad produced by S. spinosa YJY-12 demonstrated remarkable efficacy in controlling Eggplant Thrips, surpassing the control (a technical spinosad and commercial spinosad preparations). This study provides a methodological foundation for enhancing spinosad fermentation efficiency and offers guidance for future spinosad fermentation optimization research.

Materials and methods

Materials and medium

Spinosad A/D (> 94%) was purchased from QILU Pharmaceutical (Inner Mongolia) Co., Ltd. The commercial spinosad preparation was purchased from Shenzhen Noposion International Investment Co., Ltd. Glucose, methyl oleate, soybean oil, cottonseed meal, casein peptone, glycerol, sodium citrate and other reagents were commercially available.

The solid medium: glucose 7 g/L, cerealose 10 g/L, casein peptone 3 g/L, yeast extract 5 g/L, MgSO₄ 2 g/L, agar 20 g/L, pH 6.9–7.3.

The liquid seed medium: glucose 10 g/L, cerealose 10 g/L, yeast extract 20 g/L, peptone 10 g/L, soybean cake powder 15 g/L, KH₂PO₄ 3 g/L, NaCl 5 g/L, MgSO₄ 4 g/L, pH 6.9–7.3.

The seed tank culture medium: starch 15 g/L, cerealose 15 g/L, cottonseed meal 10 g/L, soybean meal 10 g/L, yeast extract 5 g/L, serine 1.5 g/L, MgSO₄ 1 g/L, (NH₄)₂SO₄ 1 g/L, CaCO₃ 2 g/L, pH 6.9–7.3.

The initial fermentation medium: glucose 50 g/L, glycerol 10 g/L, methyl oleate 30 g/L, soybean oil 15 g/L, sodium citrate 4 g/L, casein peptone 10 g/L, yeast extract 5 g/L, cottonseed meal 30 g/L, (NH₄)₂SO₄ 2 g/L, serine 1.5 g/L, KH₂PO₄ 2 g/L, FeSO₄ 0.05 g/L, CuSO₄ 0.05 g/L, NaCl 2.5 g/L, CoCl₂ 0.04 g/L, CaCO₃ 5 g/L, pH 7.4–7.5.

Supplemented medium: glucose 800 g/L.

Characterization based on 16S rRNA gene sequencing and phylogenetic analysis

Strain YJY-12 was isolated from soil samples collected from Shandong Province, and the isolation process was detailed in the supplementary materials. For the molecular characterization, the 16S rRNA gene was cloned and analyzed. The isolated genomic DNA of the microorganism was extracted by previously described³⁵ and used as PCR (Polymerase chain reaction) template. Primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACG ACTT-3′) were used for PCR amplification. The amplified PCR products were sequenced by Sangon Biotech (Shanghai, China).

The obtained 16S rRNA gene sequences were aligned and affiliations were deduced via BLAST analysis. A phylogenetic tree (neighbor-joining) was constructed by using MEGA 11.0 from dissimilar distances and distances were calculated via the Kimura two-parameter distance model²⁶.

Analysis of inheritance and ferment stability of S. spinosa YJY-12

The strain YJY-12 underwent successive subculturing for a total of five passages. The fermentation capability of spinosad produced by each subsequent generation of S. spinosa YJY-12 was evaluated by shake flask fermentation to determined the production levels.

Optimization of the fermentation conditions of S. spinosa YJY-12

According to the reported literature of spinosad fermentation by S. spinosa, nine different kinds of fermentation medium were selected for screening the initial fermentation medium, and the composition of mediums were shown in table S2. The culture conditions were as follows: bottling capacity 50 mL/500 mL, rotation speed 220 rpm, culture temperature 30 °C, inoculum volume 10% (v/v) and culture time 10 d.

Then the physical factors of culture conditions, such as seedage (24–84 h), inoculation volume (2.5–15%, v/v), fermentation temperature(26–34 °C), initial pH (6.0–8.0) of medium and culture time, were studied.

In order to improve the fermentation level of spinosad by S. spinosa YJY-12, the composition of the medium was optimized in sequence by PB experiment, steepest ascent experiment and central composite design method.

The main ingredient of initial fermentation medium, such as glucose, glycerol, methyl oleate, soybean oil, casein peptone, yeast extract, cottonseed meal, corn gluten meal and corn steep liquor powder were selected for the PB design. Each factors were selected at high and low level and the design was shown in table S3.

Based on the PB experimental results, the four factors including glucose, methyl oleate, cottonseed meal and casein peptone, were selected for steepest ascent experiment. The steepest ascent experiment design was shown in table S4.

The central composite design method was used to optimize the composition of culture medium. Taking fermentation level of spinosad as response value, glucose (A), methyl oleate (B) and cottonseed meal (C) as independent variables, the experimental design was shown in the table S5.

Fed-Batch fermentation

The cryopreservation tube from − 80 °C refrigerator was removed and inoculated into solid medium at 30 °C for 8 days to form spores. The spores were scraped and prepared spore suspension with 1–1.5 × 10⁸ cells/mL. Then, the spore suspension was inoculated into liquid seed medium with inoculation volume 1% (v/v) at 30 °C and 220 rpm for 4 days to obtain the liquid seed. The liquid seed was inoculated into fermentor with seed tank culture medium, the inoculation volume was 10% (v/v) and the incubation temperature was set at 30 °C. The dissolved oxygen of seed tank was controlled at over 30% by adjusting the rotation speed and ventilation rate, and the culture time is about 30–40 h.

The 30 L fermentor was operated at 60% of its total volume, containing 18 L fermentation medium. The seed liquid was inoculate into fermentation medium at a volume of 10% (v/v), and the culture temperature was set at 30 °C. The dissolved oxygen (DO) of fermentor was controlled at over 30% by dynamic adjustment of rotation speed and ventilation rate. Initially, the rotation speed and ventilation rate were set at 100 rpm and 1 vvm (18 L/min), respectively. Subsequently, these parameters were adjusted in response to real-time DO monitoring: the rotation speed was increased by 50 rpm increments and the ventilation rate by 0.5 vvm increments when the DO level fell below 30%, with maximum values of 600 rpm and 3 vvm (54 L/min), respectively. The pH of fermentation tank was controlled within the range of 6.5–6.7 by aqueous ammonia and 10% (v/v) sulfuric acid. The reducing sugar was maintained between 12–20 g/L by adding 80% (w/v) glucose. When the fermentation level of spinosad did not increase or increased slowly, the fermentation was ended.

Experimental study on the efficacy of spinosad in controlling Eggplant Thrips

The fermentation broth of S. spinosa YJY-12 was processed by solid–liquid separation, methanol extraction, concentration, sec-butyl acetate extraction, tartaric acid back-extraction, alkali precipitation, methanol redissolution, and recrystallization to obtain technical spinosad (detail in supplementary material). The technical spinosad, from S. spinosa YJY-12 and purchased from QILU Pharmaceutical (Inner Mongolia) Co. Ltd., was formulated into an 80 g/L insecticide. The spinosad formulations were water-based suspensions. The technical spinosad, along with phosphate and carboxylate dispersants, thickener, and water, was thoroughly mixed. The mixture was then subjected to homogenization by sand mil to achieve a particle size of 5.0 μm prior to discharge, resulting in the desired spinosad preparation. Moreover, the commercial spinosad preparation was purchased from Shenzhen Noposion International Investment Co., Ltd as a control.

Field experiments on the efficacy of spinosad in controlling Eggplant Thrips was investigated. During the peak of pest infestation, the insecticides were diluted with water and sprayed evenly. Samples sprayed with water were used as a control. Then the pesticide were sprayed from a low concentration to high concentration. Finally the control pesticide was sprayed. After changing insecticides, the sprayer was cleaned. The pesticide usage was 750 L/hm² and the dosage of the preparation was 10 g/15 L and 15 g/15 L respectively. The number of thrips was investigated before spraying pesticide, and once 1 day, 3 days and 7 days after spraying pesticide. The number of living thrips on the four leaves in the middle and upper part of the plant were counted and the leaves were usually checked in the morning when the adult insects were less active.

The efficacy of spinosad in controlling Eggplant Thrips was calculated according to the following formula:

1

E—Efficacy of Thrip control/%; PT₀—Pesticide treated area before treatment; PT₁—Pesticide treated area before treatment; CK₀—Control area before treatment; CK₁—Control area before treatment.

Analysis method

Determination of the fermentation level of spinosad

One milliliter of fermentation broth was mixed with 4 mL of acetonitrile by vibration for 30 min. Then, the mixture was centrifuged by 10,000 × g for 10 min. The fermentation level of spinosad was determined by HPLC. The quantitative HPLC was accomplished with C₁₈ reverse phase column (250 mm × 4.0 mm, 5 μm). The flow rate was set at 1.0 mL/min. Samples were eluted with a mobile phase which was consisted of acetonitrile/methanol/0.05% (w/v) aqueous ammonium acetate (45: 45: 10) and the eluent was monitored at 250 nm.

Measurement of biomass

Five milliliter culture was centrifuged at 3000 × g for 10 min to collect the wet mycelia and the proportion of the volume of wet mycelia in the culture was determined. In the industrial fermentation production process of spinosad, the volume of wet mycelia was usually detected for regulating and controlling the fermentation process. Compared to detecting dry mycelia, this method was simple and fast to detect, and could achieve real-time online control of fermentation process.

Determination of reducing sugar

The reducing sugar content in fermentation broth was determined by DNS assay³⁶.

Author contributions

Xiufeng Long and Jiewei Tian designed the experiments. Xiufeng Long and Yanrui Ma performed experiments and data analysis. Xiufeng Long, Guangpeng Liu and Jiewei Tian wrote the manuscript with valuable inputs from all co-authors.

Data availability

The 16S rRNA gene sequence of strain YJY-12 was submitted to GenBank with accession number: PP998559.

Declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-09977-x.