HU Zelin, PAN Zhaoyang, ZHAO Tianyu, WANG Yongzhen, SUN Jianan, *,and MAO Xiangzhao, 2), *

Biotransformation of Shrimp Wastes byOKF04 and Evaluation of Growth Promoting Effect in Crop Planting

HU Zelin1), PAN Zhaoyang1), ZHAO Tianyu1), WANG Yongzhen1), SUN Jianan1), *,and MAO Xiangzhao1), 2), *

1),,266003,2),,266237,

In this study, we proposed a reliable and sustainable technique for the clean utilization of shrimp wastes, which can yield a solid inoculant ofOKF04 containing micronutrients at low cost without the risk of contamination. Study of the cul- ture conditions revealed that the head of shrimpand the wheat bran acted as suitable substrates for the growth ofOKF04. With 60% initial moisture content, 30℃ culture temperature, and 5 % inoculation amount, followed by 48 hours of fermentation and 0.5% soluble starch added during the drying process (50℃ for 6 h), a solidOKF04 inoculant with a spore amount of 2.4×1010CFU g?1and a high amino acid content was obtained. The solidOKF04 inoculant was applied to cultivate pakchoi under pot experiment. As the result, of adding to, the size of stems and leaves, nutritional composition, and physio- logical activity of pakchoi were significantly (< 0.05) enhanced by solidOKF04 inoculant.OKF04 also signi- ficantly (< 0.05) increased the soil’s nutrient content and improved its microbial composition. Furthermore, pakchoi cultivated with a low dose of solidOKF04 inoculant (0.05 g kg?1soil) resulted in the best results. This study provides a new method for the preparation of microbial inoculants with solid waste shrimp heads.

shrimp wastes;OKF04; inoculant; solid state fermentation; crop growth promotion

1 Introduction

Since 2020, global shrimp production has reached 4 mil- lion tons, with Asia contributing more than 3 million tons (Shinn, 2018). In the process of shrimp processing, shrimp heads, shells, and other undesirable parts are re- moved (Kumar and Suresh, 2014). By using modern biotechnology, these wastes can be turned into high-value-add- ed products that can potentially serve as valuable biologi- cal resources. In some cases, shrimp waste is directly dried and used as a low-quality roughage for aquaculture or live- stock feed (Mathew, 2020). Amar (2006) showed that growth performance and survival ofwere improved when chitinolytic and proteolytic/non- proteolytic strains were mixed with ferment shrimp heads in the feed. Shrimp waste is also used to extract various sub- stances. Mao (2017) and Mathew (2020) reviewed the pro- gress of chemical and biological extractions of chitins, pro- teins, lipids, astaxanthin, flavor compounds and calcium car- bonate from shrimp waste. However, the energy consump-tion, cost and low extraction rate of the extraction process make it difficult to effectively use the raw materials. There- fore, we need to develop a more economical and environ- mentally friendly method to make high-value products from shrimp waste.

Due to its low wastewater generation and low energy con- sumption, solid state fermentation (SSF) has gained popu- larity as a green and sustainable solution for solid waste ma- nagement. Solid state fermentation has been widely used in the production of enzymes, organic acids, secondary meta- bolites, polyglutamate, biofertilizers, biopesticides, biosur- factants, aroma compounds, animal feed, pigments, vitamins, antibiotics, biological control agents and biofuels (Bhargav, 2008; Abu Yazid, 2017). Biowaste was used to produce biopesticide through solid-state fermentation of digestating at pilot scale (Rodriguez, 2019). Based on the many advantages of solid-state fermentation in treat- ing biological wastes, we attempted to develop a microbial inoculant for the high-value treatment of shrimp waste.

Many types of commercial microbial inoculants have been successfully applied to replace fertilizers, pesticides, and antibiotics (Trabelsi and Mhamdi, 2013). In the plant industry, microbial inoculants are widely used due to their properties such as reducing the toxicity of heavy metals, promoting nitrogen fixation, promoting the use of nutrients, synthesizing plant hormones such as indole acetic acid (IAA), and resisting various pathogens (Babalola and Glick, 2012). In addition, microbial inoculants can be applied during bio- degradation to protect the environment, such as degrading residual herbicides in the soil (Wang, 2018), degrad- ing pollutants such as organics and ammonia produced in papermaking, printing and dyeing, oil refining and high- salt wastewater (Tang, 2012) and compost (Nair and Okamitsu, 2010).

is one of the most widely used micro- bial inoculant since it can produce a variety of active sub- stances, has good environmental tolerance, and has broad antibacterial activity. By producing antimicrobial substances, competing with nutrients and growth space, and inducing systemic resistance,can be used as a biological control agent (BCAS) to protect plants from soil diseases (Saxena, 2020). In our previous work (Sun, 2015), we isolated a highly active probiotic strain,OKF04, from deep-sea mud. Our preliminary studies indicate that this strain is suitable for submerged fermenta- tion using Antarctic krill meal as a carbon source and ni- trogen source to yield a high-quality fermentation broth con- taining bioactive compounds. Thus, we attempted to create a microbial inoculant by solid state fermentation of shrimp shell powder usingOKF04. Until now, there has not been any report on the production ofby so- lid-state fermentation on shrimp wastes.

In this study, we aim to use solid-state fermentation to treat shrimp processing waste in a way with low-cost, low- energy, and low-polluting in order to obtain solid probioticOKF04. We present a method for the treatment of shrimp waste, the preparation ofOKF04 in- oculant using solid-state fermentation, thus overcoming the shortcomings of submerged fermentation with high energy consumption and waste water. At the same time, the biolo- gical activity ofOKF04 was preliminarily veri- fied through pot experiment.

2 Materials and Methods

2.1 Strains and Substrates

TheOKF04 strain was isolated from deep sea mud (?1137 m, 126?38.61?E, 27?49.23?N) by using Antarc- tic krill power as the sole carbon and nitrogen source and preserved in the laboratory (Sun, 2015). Substrates of solid state fermentation shrimp () head and wheat bran were purchased from Qingdao Sheng- long Weiye Technology Co., Ltd.

2.2 Preparation of Bacillus subtilis Inoculant by Solid State Fermentation

The strain ofOKF04 stored at ?20℃ in gly- cerol was inoculated into Luria-Bertani substrate with 1% inoculation amount, then cultured in shaking flask at 200 r min?1at 30℃ for 24 h for activation. In the substrate of glu- cose 30.0 g L?1, KH2PO41.0 g L?1, yeast powder 25.0 g L?1, NaCl 0.5 g L?1, MgSO4·7H2O 0.5 g L?1, the initial pH 7.0, liquid volume 30 mL (250 mL shaking flask), inoculation volume 8% (V/V), culture temperature 28℃ for high den- sity culture, the solution with 6.7×109CFU mL?1OKF04 was obtained.

After culturingOKF04 at high density, it was subjected to solid-state fermentation in shrimp head solid medium. Then the biomass ofOKF04 in solid state fermentation with different moisture content (40%, 50%, 60%, 70%, 80%), fermentation temperature (28, 30, 34, 37℃), inoculation amount (2%, 5%, 10%, 15%), amounts of protectant (0, 0.5%, 1%, 2%, 3%, 4%, 5%) and drying time (0, 2, 4, 6, 8, 10, 12 h) were determined. After- wards, the inoculant was observed with a scanning electron microscope (SEM) of TESCAN VEGA3.Totally 1 g of un- fermented sample and inoculant were taken, respectively, and they were sticked on a copper sheet with double-sided tape and were placed on the specimen table. The samples were placed in the sputter coater and plated. They were scanned at 5000 and 10000 times magnification.

2.3 Determination of Inoculum Biomass

For preparation of a leaching solution for solid inoculant, 2 g solid sample was added into 20 mL sterile water con- taining 7 – 10 glass beads and placed in a 200 r min?1ther- mostatic oscillator for 30 min to obtain the leaching solu- tion. The amount ofcolony forming units was determined by plate count. To prepare 1:10 sample solution, 100 μL leaching solution was added to 900 μL sterile sa- line, then was mixed by vortexing. To do 10 times gradient dilution, 100 μL diluent of each dilution was injected into the count plate, the diluent was evenly coated with stick and then placed upside down. The count plate was incubated in incubator at 30℃ for 24 h. Plates with 30 to 300 colonies were selected to count the numbers of the colonies. Spores colony forming units were counted when the leaching so- lution was bathed in water at 80℃ for 15 min.

2.4 Determination of Free Amino Acid Content

The free amino acid content was determined by S433D amino acid analyzer (SYKAM GmbH, Germany). The ex- periments were performed on a high-efficiency sodium ca- tion-exchange Pickering Laboratories column (4.0 mm × 150 mm). It was operated using a mobile phase of lithium citrate with pH 2.9, pH 4.2, and pH 8.0, respectively. UV- vis detection was performed at 570 nm with the Sykam S433D Physiological Li C4 system. In this experiment, the mobile phase flowed at 0.45 mL min?1and the derivatizing reagent flowed at 0.25 mL min?1. The column temperature was set at 48℃, and the post-column reaction equipment was kept at 130℃. The temperature of the autosampler was kept at 5℃, and the injection volume was 10 μL for both standard and experimental samples (Sun, 2014). The amino acids included aspartic acid, threonine, serine, gluta- mic acid, glycine, alanine, valine, methionine, proline, iso- leucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, cysteine, while the total amino acid content was also determined.

2.5 Potted Plant Experiment Materials

The size of the potted greenhouses is 180 cm × 124 cm × 70 cm, and the size of the cultivation pots is 39 cm × 29 cm × 15 cm. The experimental crop is pakchoi (Linn.). Seeds for pakchoi were purchased from Shou- guang Xinxin Horticulture Co., Ltd. Our cultivated soil (pH 5.53, organic matter 25.9%, hydrolyzed nitrogen 921 mg kg?1, available phosphorus 58.8 mg kg?1, and exchange- able potassium 565 mg kg?1) was purchased from Shouguang Jinguo Agricultural Science and Technology Co., Ltd. The cultivation site is located at the Yushan Campus of Ocean University of China (120?20?6.26??E, 36?3?38.66??N).

2.6 Potted Plant Experiment Design

The planting experiment was carried out in a greenhouse. 4 kg of soil were added to each pot and a total of 4 groups were set up: blank control group (CK), unfermented sam- ple (0.2 g per 4 kg soil) treatment group (UF), low-doseOKF04 inoculant (0.2 g per 4 kg soil) treatment group (FD), and high-doseOKF04 inoculant (0.8 g per 4 kg soil) group (FG). Three parallel experiments were conducted in each group. Healthy pakchoi seeds were se- lected, 40 seeds were added to each pot for planting, and 24 seedlings were kept in each pot after the leaves grew out. After 40 days, relevant indexes were measured.

2.7 Pakchoi Growth Morphological Parameters

Plant height, root length, maximum leaf width, maximum leaf length, fresh weight of stem and leaf, and fresh weight of root were measured directly. Plant height is the distance from the rhizome to the peak of leaf growth. The stem, leaf, and root were cured at 105℃ for 30 min and then dried at 75℃ for 24 h to determine the dry weight.

2.8 Pakchoi Physiological Parameters

Leaves of pakchoi were removed after 40 days of plant- ing and vitamin C in the leaves was determined using the fluorimetric method (F-4600, HITACHI, Japan) according to the method of Chung (1991): 10 g of leaves was added to 100 g of metaphosphoric acid-acetic acid solution (weigh 15 g metaphosphoric acid and add 40 mL of glacial acetic acid and 250 mL of water, then add 500 mL of water af- ter cooling) and homogenized with mash machine (BILON- J1500, BILON, China). The pH of the sample solution was adjusted to 1.2 using metaphosphoric acid-acetic acid so- lution and metaphosphoric acid-acetic acid-sulfuric acid so- lution (weigh 15 g of metaphosphoric acid, add 4 mL of gla- cial acetic acid, add 0.15 mol L?1sulfuric acid solution drop- wise until dissolved and dilute to 500 mL). Then 1 mL of the sample solution was diluted to 100 mL. The diluted sam- ple solution was pipetted into a 200 mL conical flask along with 2 g activated carbon, and was shaken for 1 min. Then they were filtered to obtain the oxidising solution. Accu- rately 10 mL of the oxidising solution was transferred into two 100 mL volumetric flasks respectively. Then 5 mL of boric acid-sodium acetate solution (3 g of boric acid was dis- solved with 500 g L?1sodium acetate solution and dilute to 100 mL) and 500 g L?1sodium acetate solution was mixed, and was diluted to 100 mL respectively. Then 2 mL of the above samples were added into 10 mL stoppered graduated tubes, 5 mL of 200 μg L?1o-phenylenediamine solution was added into each tube rapidly in the dark room, mixed with shaking, and the reaction was carried out at room tempe- rature for 35 min. The fluorescence intensity was measured at excitation wavelength 338 nm and emission wavelength 420 nm.

Chlorophyll content in the leaves was determined accord- ing to the method of Arnon (1949). The sample was weigh- ed 0.2 g, placed in a 100 mL glass mortar, 0.2 g of calcium carbonate and 0.5 g of quartz sand were added and ground for 5 min, then 2 mL of acetone-water solution (85% ace- tone, 15% water) was added and grinding was continued un- til it became a dry powder. All grinds were transferred to a G3glass funnel, slowly filtered, washed repeatedly with acetone-water solution, and filtered until the filtrate was al- most colorless. All the filtrate was transferred to a 250 mL separatory funnel containing 50 mL of 2% sodium sulfate solution, the aqueous phase was transferred to another se- paratory funnel, 10 mL of ether was added for extraction, and the aqueous phase was discarded. The organic phase was incorporated into the first partition funnel and washed twice with water, 20 mL each time, and the aqueous phase was dis- carded. The filtrate was prepared by filtering the ether phase through a funnel containing 5 g of anhydrous sodium sul- fate into a 50 mL brown conical flask. Then 15 mL of the fil- trate was taken and fixed with ether to 50 mL, and the ab- sorbance values were measured at 642 nm and 660 nm us- ing a spectrophotometer (UV-5800, METASH, China), with ether as a blank control.

Root activity was determined by the triphenyltetrazolium chloride method (TTC) (Liu and Zhang, 2022). In this ex- periment, the root activity was defined as the weight of 1 g of root reducing triphenyltetrazolium chloride (TTC) to tri- phenylformazan (TTF) in 1 h. A sample of 0.5 g of clean- ed roots was weighed and added to a 10 mL brown reagent bottle with 5 mL, 0.4% TTC solution and 5 mL, 0.1 mol L?1phosphate buffer. The roots were fully submerged in the solution and stored at 37℃ for 2 h protected from light. At the end of the reaction, the roots were placed in a mortar and 2 mL of ethyl acetate was added and ground thorough- ly. The TTF solution was transferred to a 10 mL cuvette. Finally, the absorbance was measured at 485 nm.

The determination of enzymatic activity of leaves was carried out using superoxide dismutase (SOD) activity as- say kit (Solarbio LIFE SCIENCES, China) and catalase (CAT) activity assay kit (Solarbio LIFE SCIENCES, Chi- na).

2.9 Soil Nutrients Parameters

Totally 5 g soil sample from the depth 0 – 10 cm was se- lected to determine the contents of soil nutrients (organic matter, hydrolyzed nitrogen, available phosphorus, exchange- able potassium). The soil organic matter was determined by K2Cr2O7-H2SO4oxidation method following Walkey and Black (1934). The soil sample of 0.02 g was taken, 0.1 g of silver sulfate was added, and then 10 mL, 0.4 mol L?1of po- tassium dichromate-sulfuric acid solution was added and digested in a digestion tube for 5 min. Then, 4 drops of o- phenanthroline were added and the remaining potassium dichromate was titrated with ferrous sulfate standard solu- tion (56 g of ferrous sulfate dissolved in 700 mL of water, then 20 mL of concentrated sulfuric acid, and then fixed to 1 L).

Hydrolytic nitrogen in soils was determined using the alkaline diffusion method (Lu, 2011). 2 g of air-dried soil was placed in the outer chamber of the diffusion dish. Then 0.2 g of ferrous sulfate and 2 mL, 20 g L?1of boric acid in- dicator were added to the inner chamber of the diffusion dish, then the edge of the outer chamber of the dish was coated with alkaline gum solution, covered with hairy glass, and 10 mL, 1 mol L?1of sodium hydroxide solution was add- ed to the outer chamber of the diffusion dish. Then, the dif- fusion dish was placed into a constant temperature incuba- tor (DHP-9211, Yiheng, China) at 40℃ and removed after 24 h of alkaline diffusion. The liquefied NH3absorbed in the inner chamber was titrated with 0.01 mol L?1sulfuric acid standard solution.

Available phosphorus in soils was determined using Ol- sen’s method (1954). The air-dried sample was weighed 5 g in a 200 mL plastic bottle, 50 mL of ammonium fluoride- hydrochloric acid solution was added (1.11 g of ammonium fluoride dissolved in 400 mL of water, 2.1 mL of hydrochlo- ric acid was added and diluted to 4 L with water) and shaken for 30 min. The above solution was filtered through filter paper to produce the extracting solution. The 10 mL filtrate was aspirated into a 50 mL volumetric flask, then 10 mL, 30 g L?1of boric acid solution (30 g L?1) was added, and wa- ter was added to 30 mL. Then two drops of dinitrophenol indicator were added to the volumetric flask, followed by 5 mL of Mo-Sb colorimetric solution, and the volume was fixed to 50 mL with water. The absorbance was measured at 700 nm using a spectrophotometer (UV-5800, METASH, China).

Exchangeable potassium in soil was measured with a flame photometer (AA-6800, Shimadzu, Japan) as described by Jackson (1973). 5 g of air-dried soil sample was placed in a 200 mL plastic bottle, and then 50 mL, 1 mol L?1am- monium acetate solution was added and shaken for 20 min at 25℃, and then the filtrate was filtered to obtain the fil- trate. The filtrate was measured directly on a flame photo- meter.

2.10 Number of Soil Microorganisms

Totally 10 g soil sample from 0 – 10 cm was collected to determine the number of microorganisms. The contents of fungi, bacteria, and actinomycetes in soil were measured with the same way as that of 2.3. Bacteria were cultured with Beef paste protein agar medium, fungi with Bengal red medium, and actinomycetes with Gao’s No. 1 medium.

2.11 Soil Enzyme Activity Parameters

Totally 5 g soil sample from 0 – 10 cm was collected to determine the enzymes activities. Soil urease (S-UE) acti- vity test kit (Solarbio LIFE SCIENCES, China), soil su- crase (S-SC) activity test kit (Solarbio LIFE SCIENCES, China), soil alkaline phosphatase (S-AKP/ALP) activity test kit (Solarbio Life Sciences, China), and soil neutral pho- sphatase (S-NP) activity test kit (Solarbio Life Sciences, China) were used following the instructions of the manu- factures, respectively.

2.12 Statistical Analysis

Statistical differences of the results were analyzed us- ing a one-way ANOVA with the Tukey test. Assays were conducted with triplicates, and the values were reported as mean value ± standard deviation (SD). SPSS (IBM SPSS Sta- tistics 25) was used to analyze the data. It was considered as significant when< 0.05.

3 Results and Discussion

3.1 Biomass of Solid B. subtilis OKF04 Inoculant

To prepareOKF04 inoculant with a high viable count, we optimized the production process ofOKF04 inoculant by using solid-state fermentation and mea- sured its biomass. During the solid-state fermentation, the viable count content ofOKF04 inoculant in- creased and reached the maximum value at the 48th hour, and then gradually decreased (Figs.1A, B, C). TheOKF04 inoculant had the highest viable count content when the initial moisture content was 60%. The optimum moisture content for solid state fermentation varies depend- ing on the type of substrate, and generally it is between 50% and 70%. When the substrate has absorbed enough water, it is well suited for inoculation and fermentation (Chen, 2020). The results of this study showed that when the moisture content of the substrate is 80%, some water will seep out. When the moisture content is 40%, the substrate is not completely wet. Both of them can inhibit the growth of bacteria. When the moisture content is 60%, the substrate is completely wet without any water seepage. Consequently, the optimal moisture content for solid-state fermentation ofOKF04 is 60%.

TheOKF04 inoculant had the highest viable count content when the incubation temperature was 30℃. With an increase in fermentation temperature, the num- ber of viableOKF04 decreases faster with an in- crease in fermentation time (Fig.1B). A majority of solid state fermentation studies usingfocus on tempe- ratures between 30 and 40℃, with 36℃ being the optimal temperature. However, these studies tend to focus on the extracted products (Niu, 2015; Chen, 2020; Li and Wang, 2021). The main objective of our study is to determine the amount of living bacteria. Therefore, 30℃ is the optimal temperature forOKF04 during so- lid-state fermentation.

TheOKF04 inoculant had the highest viable count content when the inoculation amount was 5% or 10%. Too much inoculation amount can lead to a large amount of heat accumulation, which severely reduces the number of viable bacteria. Moreover, high inoculation amount can also increase costs. Therefore, fermentation conditions with a low inoculation amount should be selected. The optimal inocu- lation amount forOKF04 is determined to be 5%.

In order to improve the survival rate ofOKF04 during the drying process, we optimized the type and ad- dition of protective agents. From the results, trehalose had the highest protective effect with a survival rate of 60.89%, soluble starch had the second best protective effect with a survival rate of 49.67%, while the survival rate when solu- ble starch was added was not significantly (> 0.05) dif- ferent compared with trehalose (Fig.1D). We added soluble starch with a concentration of 0.5% – 4% to the inoculant during the drying process, and the survival rate ofOKF04 increased significantly (< 0.05) over that of the inoculant without soluble starch, and 85.71% was the highest survival rate when soluble starch was added at 0.5% (Fig.1E). During the drying process, water loss in the cell membrane will destroy hydrogen bonds between phospho- lipids, proteins, and water molecules. As the result, the cell membrane will not function properly (Niu, 2015). To maintain the structure of the lipid bilayer of the cell mem- brane, trehalose can be substituted for water molecules. Ad- ditionally, trehalose has a high glass transition temperature, it can remain glassy longer than other nonreducing disac- charides (such as sucrose). As a result of the glassy state with high viscosity, the cells are provided with additional protection (Fu and Chen, 2011). As well as trehalose, solu- ble starch may also form a glassy state when dried, which can strongly adhere to the cells and maintain the activity of proteins and cell membranes, thus improving cell viability (Trongsatitkul, 2019). Additionally, the cost of solu- ble starch is lower than that of trehalose. Therefore, we added 0.5% soluble starch to the drying process. Addition- ally, we determined the drying time of the inoculant. Af- ter 6 hours of drying, the moisture content of the inocu- lant was 7% and spore counts reached the highest level at 2.4 × 1010CFU g?1. Therefore, 6 hours was the most suitable time for the preparation of the inoculant (Fig.1F).

Fig.1 The amount of B. subtilis OKF04 inoculant colonies forming with different moisture content (A), fermentation tem- perature (B), inoculation amount (C), protectant type (D), protectant amount (E), and drying time (F).

3.2 Free Amino Acids Content of the B. subtilis OKF04 Inoculant

The results shown in Table 1 demonstrate that the free amino acids’ contents in the solid bacterial agent was sig- nificantly higher than those of the unfermented control (< 0.05). Among the 17 kinds of free amino acids we mea- sured, the contents of glutamic acid, aspartic acid, serine, valine, methionine in the solid bacterial agent was signifi- cantly higher than those of the unfermented control (< 0.05).contains a large number of proteases, which can break down the proteins in the substrate into free amino acids and small peptides during the fermentation process (Wang, 2014; Harwood and Kikuchi, 2022). Nume- rous studies have demonstrated that free amino acids are directly absorbed by plant roots and play an important role in improving soil ecology and plant growth (Schobert, 1988; N?sholm, 2009). Among these free amino acids, aspartic acid, glutamic acid and methionine were elevated to a large extent by 29%, 10% and 219%, respectively. Glu- tamic acid and aspartic acid are the main amino acids in- volved in the process of translocation from the roots to the shoot of plants and have a growth-promoting effect (For- sum, 2008). The above results indicate thatOKF04 inoculant is rich in free amino acids compared to the unfermented control. TheOKF04 inocu- lant has the potential to be used in the cultivation of crops.

Table 1 Contents of free amino acid of unfermented control and solid inoculant

3.3 Micromorphology of the B. subtilis OKF04 Inoculant

Scanning electron microscopy (SEM) was used to mea- sure the inoculant prepared by the solid-state fermentation method as well as the substrate without fermentation. The results are shown in Fig.2. The inoculant (Figs.2B and D) attaches a greater number of rod-shaped bacteria than the unfermented substrate (Figs.2A and C). Accordingly,OKF04 could be used as a carrier for fermenting shrimp waste. There are a few globules visible in Fig.2B, which may be the result of bacterial contamination during the placement of the sample.

3.4 Pakchoi Growth Morphological Indexes and Nutrient Content

Growth morphological index and chlorophyll content, at leaf and canopy level, are important variables for agricul- tural applications because of their crucial role in photosyn- thesis and in plant functioning (Clevers, 2017). The morphological indexes of growth of each group of pakchoi were determined with different levels of inoculant added. According to Table 2, compared with CK, there was no sig- nificant difference (> 0.05) in all growth morphological in- dicators of UF-treated pakchoi, while the maximum leaf length, maximum leaf width, plant height, root length, stem and leaf fresh weight, stem and leaf dry weight, root fresh weight, and root dry weight of FD-treated pakchoi increased by 25%, 19%, 37%, 33%, 38%, 45%, 9%, and 32%, respec- tively. The maximum leaf length, leaf width and plant height of FD-treated pakchoi were significantly higher than those of the CK, UF and FG groups (< 0.05). As shown above, the low dose ofOKF04 inoculant was the most suitable for the growth of pakchoi.

Fig.2 Scanning electron microscope image of solid Bacillus subtilis OKF04 inoculant. A, unfermented substrate (5000×); B, solid inoculant (5000×); C, unfermented substrate (10000×); D, solid inoculant (100000×).

Crop growth and yield are indirectly related to chloro- phyll content, which can be regarded as a reflection of pho- tosynthetic capacity (Wang, 2021). We determined the chlorophyll content, vitamin C content and root activity of pakchoi leaves (Table 2). Compared with CK, chlorophyll content did not change much under UF treatment, but it in- creased significantly (< 0.05) under FD and FG treatments, which increased by 49% and 57% respectively. Among them, the highest chlorophyll content was found under FG treatment. Compared with CK, UF, FD and FG treatments all increased the vitamin C content in the leaves. The re- sults showed that 1.3 times more vitamin C was present in the UF-treated leaves than in the CK group, while twice as much vitamin C was present in the FD- and FG-treated leaves than in the CK group. It is also believed that the plant’s high vitamin C content contributes to its antioxidant properties. In a study conducted by Liang (2022),Ydj3 was used for the cultivation of bell pepper. Vita- min C content of treated plants (790.9 μg g?1± 17.9 μg g?1) was significantly (< 0.05) higher than that of untreated control plants (730.8 μg g?1± 14.4 μg g?1). Root activity was significantly (< 0.05) higher in the FD and FG treatments compared to the CK treatment, which incrased by 26% and 41%, respectively, while in the UF treatment there was no significant difference (> 0.05). The above results indicated thatOKF04 inoculant could increase the nutri- ent content and root activity of pakchoi leaves. This indi- cates thatOKF04 inoculant can be used as a fine microorganism fertilizer in crop cultivation.

Table 2 Growth morphological indexes and nutrient content of pakchoi with different treatment

Notes: CK, blank control group; UF, unfermented sample (0.2 g per 4 kg soil) treatment group; FD, low-doseOKF04 inoculant (0.2 g per 4 kg soil) treatment group; FG, high-doseOKF04 inoculant (0.8 g per 4 kg soil) group.*Values in columns with different superscript letters are significantly different according to Tukey’s multiple range test (< 0.05).

3.5 Effect of B. subtilis OKF04 Inoculant on Soil Nutrients

According to Table 3, compared to CK, hydrolyzed nitro- gen, available phosphorus, and exchangeable potassium con- tents were significantly (< 0.05) increased in UF treatment by 10%, 39%, and 20%, respectively. In FD treatment, soil organic matter, hydrolyzed nitrogen, available phosphorus, and exchangeable potassium were significantly (< 0.05) higher in the FD treatment, with increases of 10%, 15%, 59%, and 31%, respectively. In the FG treatment, soil orga- nic matter, hydrolyzed nitrogen, available phosphorus, and exchangeable potassium significantly (< 0.05) increased by 5%, 29%, and 39%, respectively. The above results indi- cated thatOKF04 inoculant could increase soil nutrient content. A healthy soil is essential for crop growth since it provides the necessary nutrients and water for plant roots to develop as well as for microorganisms to thrive (Wolejko, 2020). The organic matter content of the soil, the hydrolytic nitrogen content, the effective phospho- rus content, and the exchangeable potassium content are all critical indicators for assessing soil fertility (Chen, 2018). The absorption of adequate phosphorus can promote root development, increase productivity, enhance natural defense mechanisms against plant pathogens, improve yield and quality, and sometimes lead to early crop maturation (Mikkelsen, 2007). After sufficient fermentation byOKF04, the substrate is converted into organic matter and inorganic salts that can be used by plants, which largely improves the fertility of the soil.

Table 3 Soil nutrients and microorganisms with different treatment

Notes: Same as those in Table 2.

3.6 Effect of B. subtilis OKF04 Inoculant on Soil Microorganisms

We determined the number of different species of micro- organisms in the soil. According to the results in Table 3, there was no significant difference (> 0.05) in the con- tent of soil bacteria and actinomycetes in the UF treatment compared to CK. In contrast, there was a significant (< 0.05) increase in the content of soil bacteria in FD and FG treatments, and a significant (< 0.05) decrease in the con- tent of soil fungi in FD and FG treatments. These results in- dicated thatOKF04 inoculant could improve the microbial composition of the soil. It is well known that soil microorganisms play a significant role in the degrada- tion of organic matter, energy flow, and element cycling, as well as influence soil fertility, soil enzyme activity, and plant disease resistance, which can be used as indicators of soil health (Mahdi, 2017). Microorganisms have been found to promote the mineralization of humified soil, although the number of microbes is not a key factor in the improvement of soil organic matter (Brookes, 2008). After the addition ofOKF04 inoculant, the count of bacteria and actinomycetes increased, which had an in- hibitory effect on fungi. Fungi are saprophytic microorga- nisms in the soil and can degrade macromolecules in the soil. However, the increase of fungi in the rhizosphere soil is the main cause of root rot (Shen, 2016). The de- crease of the number of fungi in the rhizosphere soil was beneficial to the growth of pakchoi, which was consistent with the change of other indicators.

3.7 Enzyme Activity Changes in Pakchoi Leaves and Soil

We determined the activity of enzymes in leaves of pak- choi and in soil. There was no significant difference (> 0.05) in SOD activity and CAT activity of leaves under UF treatment compared with CK, while SOD activity and CAT activity were significantly (< 0.05) enhanced under FD and FG treatments, with the highest SOD activity and CAT ac- tivity under FD treatment, as shown in Fig.3A. SOD and CAT are important enzymes for scavenging reactive oxy- gen species in plants and play an important role in the over- come the oxidative stress (Ulgen, 2021). A major function of SOD is to scavenge superoxide radicals and to reduce the production of hydroxyl radicals, which is achi- eved by the Huber-Weiss reaction. CAT protects plants from damage mainly by converting hydrogen peroxide (H2O2) into water (H2O) and molecular oxygen (O2) through a catabolic reaction (Bowler, 1992; Osmond, 2000). According to the results of this study, the addition ofOKF04 inoculant resulted in an increase in SOD and CAT activities of the leaves by 113% and 87%, respectively. Lee. (2015) inoculatedKFRI 1124 andKFRI 1127 on ginseng seeds, and after 24 h of fermentation, the SOD activities in the ginseng seed increased by 31.6% and 32.7%, respectively. The above changes indicated that the antioxidant capacity of pakchoi was substantially improved after the addition ofOKF04 inoculant.

As the products of soil microorganisms, animals and plants, soil enzymes participate in a variety of biochemical process- es in the soil, such as hydrolysis and transformation of or- ganic compounds, oxidation and reduction reactions of some inorganic compounds, and are an important indicator of soil changes (Burns, 2013). There was no significant dif- ference in S-NP and S-AKP/ALP activity under UF treat- ment compared with CK (> 0.05). FD and FG treatments both increased S-NP and S-AKP/ALP activity, with FD treatment having the most effective result, increasing S- NP and S-AKP/ALP activities by 19% and 30%, respective- ly (Fig.3B). Soil phosphatase activity and the decomposi- tion and mineralization of organic phosphorus elements in soil can be used as an important index to identify soil fer- tility (Parton, 1988). The soil urease activity under FD and FG treatments was significantly higher (< 0.05) than that under CK and UF treatments. Soil urease is re- lated to soil nitrogen transformation, and its activity is cor- related with the contents of total nitrogen and available ni- trogen, which is an important indicator to characterize the nitrogen supply capacity of soil (Pinto Vilar and Ikuma, 2021). Compared with CK and UF, soil sucrase activity was significantly (< 0.05) increased by FD and FG treatments. Soil sucrase can promote the hydrolysis of sucrose into glu- cose and fructose which can be easily utilized by plants and microorganisms, and plays an important role in increasing the soluble nutrients in soil. Soil sucrase activity was posi- tively correlated with soil organic matter content and total nitrogen content (Zhou, 2005), which was consistent with the higher organic matter content in FD and FG treat- ments.

Fig.3 Effects of different treatments on enzyme activities of leaves (A) and soil enzyme activity (B). The enzymes include superoxide dismutase (SOD), catalase (CAT), soil urease (S-UE), soil sucrase (S-SC), soil alkaline phosphatase (S-AKP/ ALP), soil neutral phosphatase (S-NP). The samples include blank control group (CK), unfermented sample (0.2 g per 4 kg soil) treatment group (UF), low-dose B. subtilis OKF04 inoculant (0.2 g per 4 kg soil) treatment group (FD), high-dose B. subtilis OKF04 inoculant (0.8 g per 4 kg soil) group (FG). According to Tukey’s multiple range test, multiple comparisons were per- formed for the same indicator in different groups, with different letters representing significantly different values (P < 0.05).

In summary, by means of solid-state fermentation, it was determined that under the conditions of initial mois- ture content of 60%, culture temperature of 30℃ and ino- culation amount of 5% for 48 h, there were more beneficial components and the fermentation status was stable. After fermentation, 0.5% soluble starch was added, mixed even- ly and dried at 50℃ for 6 h to prepare solidOKF04 inoculant. The product inoculant containsOKF04 spore number up to 2.4×1010CFU g?1. At the same time, the product inoculant also contains 233.6 mg g?1of free amino acids. The growth performance, soil micro- bial environment and root enzyme activity of pakchoi were improved by usingOKF04 inoculum at 0.05 g kg?1in pot experiment. TheOKF04 inoculant with wheat bran and shrimp head by solid-state fermenta- tion has a good application prospect. This study provides a reference for the preparation of microbial inoculants from solid waste shrimp head as substrate. Further research is re- quired to determine the effects of its application in breed- ing industries and to determine the amount of its applica- tion.

4 Conclusions

In this study,OKF04 inoculant was prepared by solid-state fermentation using shrimp shell waste and wheat bran as substrates, and it was applied in the cultiva- tion of pakchoi. The optimal fermentation conditions were developed. The growth performance, soil microbial envi- ronment and root enzyme activity of pakchoi were improved by culturing pakchoi with 0.05 g kg?1OKF04 in- oculant.

Acknowledgements

This study was funded by the China Agriculture Research System of MOF and MARA (No. CARS-48), and the Taishan Scholar Project of Shandong Province (No. tsqn201812020).

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