A method for enhancing the pullulan production capacity of papermaking bleaching mud from *Bacillus buddingus*.

Abstract

This invention discloses a method for enhancing the pullulan production capacity of *Bacillus buddingus* using papermaking sludge. The method involves adding papermaking sludge as an inorganic waste to a pullulan-producing culture medium, followed by fermentation of pullulan by *Bacillus buddingus*. This invention significantly enhances the pullulan production capacity of *Bacillus buddingus* by adding papermaking sludge as an inorganic waste to the pullulan-producing culture medium, while maintaining its original viscosity level, thus reducing production costs. It pioneers research on the correlation between papermaking sludge and pullulan, enriching the reduction and resource utilization of papermaking sludge. It can also be used to screen potential high-yield pullulan-producing strains, enriching germplasm resources for functional polysaccharides. It has good environmental and economic benefits.

Description

Technical fields:

[0001] This invention belongs to the field of resource utilization of papermaking waste, and specifically relates to a method for enhancing the ability of papermaking sludge to produce pullulan polysaccharide from budding short-stalked mold. Background technology:

[0002] Pullulan is a linear extracellular glucan secreted by *Aureobasidium spp.*. Pullulan possesses unique and excellent physicochemical properties, including water solubility, adhesiveness, biocompatibility, oxygen barrier properties, easy film formation, easy degradation, and flexibility. It is also rich in various functional groups, making it easy to modify, and is safe, acid and alkali resistant, and salt resistant. Therefore, pullulan and its derivatives have broad application prospects in the food and preservation, pharmaceutical, cosmetic, and environmental protection industries. Currently, domestic and international demand for it is constantly increasing. However, the pullulan market is mainly monopolized by the United States and Japan, resulting in high prices; for example, food-grade pullulan costs between 230,000 and 300,000 yuan per ton. Due to the different specificities and high osmotic pressure tolerance of *Aureobasidium spp.*, its pullulan production capacity is limited, which restricts the widespread application of pullulan.

[0003] my country is a major paper-producing country, generating approximately 50 million tons of paper annually, accounting for over 50% of global production. Papermaking sludge is a byproduct of the causticizing reaction during alkali recovery in pulp and paper mills, with a yield of approximately 0.5 million tons. 3 The annual output of papermaking sludge is enormous, with each ton of pulp producing a significant amount. Papermaking sludge primarily contains CaCO3, small amounts of CaO, and trace elements (such as Mg, K, and Fe). It is highly alkaline (pH 11–13.5) and easily harms the ecological environment. From a resource utilization perspective, current domestic and international research mainly focuses on applying papermaking sludge to building materials (such as coatings and cement), CaCO3 recovery, acid soil remediation, adsorption of heavy metals, phosphorus, and CO2, and modification for composite material preparation. However, due to insufficient technological maturity or high investment costs, the industrial utilization of papermaking sludge is limited, and traditional landfill or stockpiling is still widely used, leading to environmental pollution and resource waste. It has been reported that papermaking sludge can be used as an acidification buffer and inorganic nutrient supplement in anaerobic fermentation systems for hydrogen and biogas production from kitchen waste, significantly improving the stability and biogas production rate of the fermentation system.

[0004] High-yield pullulan production requires budding short-stem molds under conditions of high concentrations of a single carbon source (such as glucose). To reduce production costs, many researchers have used industrial and agricultural organic byproducts and starch wastewater to replace carbon or nitrogen sources in pullulan production, but the yield and efficiency are generally low. Currently, there is no precedent for utilizing inorganic waste to increase pullulan production. Based on the characteristics of papermaking sludge and its ability to improve the hydrogen and biogas production performance of anaerobic fermentation of kitchen waste, using papermaking sludge as inorganic waste for pullulan production has the advantages and potential to increase pullulan yield, reduce costs, and achieve resource utilization of papermaking sludge, thus realizing both environmental and economic benefits. Summary of the Invention:

[0005] The purpose of this invention is to provide a method for enhancing the pullulan production capacity of *Bacillus buddingus* by adding papermaking sludge as an inorganic waste to a pullulan-producing culture medium, and then utilizing *Bacillus buddingus* to produce pullulan. This method can significantly enhance the pullulan production capacity of *Bacillus buddingus* while maintaining the original viscosity level of the polysaccharide.

[0006] The present invention relates to a method for enhancing the pullulan production capacity of papermaking sludge by *Bacillus buddingus*, which involves adding papermaking sludge as an inorganic waste to a pullulan-producing culture medium, and then using *Bacillus buddingus* for fermentation to produce pullulan.

[0007] Preferably, the inorganic waste papermaking sludge is added directly to the culture medium.

[0008] The culture medium described is a conventional medium suitable for the production of pullulan by budding short-stalked fungi.

[0009] Preferably, the culture medium for producing pullulan contains, by mass fraction, 9-15% sucrose, 0.1-0.5% yeast extract, 0.2-0.8% K2HPO4, 0-0.45% NaCl, and 0.02-1.0% (NH4)2SO4, and the proportion of papermaking sludge in the culture medium is 0.1-0.5% w / v.

[0010] Further preferably, the culture medium for producing pullulan comprises, by mass fraction, 12% sucrose, 0.25% yeast extract, 0.4% K₂HPO₄, 0.05% NaCl, 0.04% (NH₄)₂SO₄, and 0.1-0.5% papermaking sludge, w / v, with the balance being water. More preferably, the proportion of papermaking sludge in the culture medium is 0.4% w / v.

[0011] The aforementioned budding short-stem mold is a short-stem mold strain with pullulan polysaccharide production function, which can be obtained through screening or purchased from a bacterial bank, and may be Aureobasidium pullulans.

[0012] Further optimization revealed that the budding short-stemmed fungus was Aureobasidium pullulans E1.

[0013] Preferably, the specific steps are as follows: 1) Cell activation: Streak the *Bacillus brevis* strain on YPD plates and incubate at 25–35°C for 2–3 days to obtain cells; 2) Obtaining seed culture: Pick cells and place them in YPD liquid medium, and incubate at 25–35°C and 180–250 rpm for 1–2 days; 3) Fermentation for sugar production: Inoculate the seed culture into a pullulan-producing medium containing papermaking mud at an inoculum volume ratio of 5–10%, and ferment at 25–35°C and 180–250 rpm for 3–5 days; 4) Detection of polysaccharides and biomass: Centrifuge the fermentation broth at 12,000 rpm for 10 min, and then precipitate the supernatant with alcohol and dry it (including the cells) to obtain polysaccharide yield and biomass, and detect the viscosity of the supernatant; The pullulan-producing medium containing papermaking mud contains, by mass fraction, 12% sucrose, 0.25% yeast extract, 0.4% K₂HPO₄, and NaCl. 0.05%, (NH4)2SO4 0.04%, papermaking sludge 0.1-0.5% w / v.

[0014] Through experiments, the inventors discovered that adding papermaking sludge to the culture medium of pullulan produced by *Bacillus buddingus* can significantly enhance its pullulan production capacity while maintaining the original viscosity level of the polysaccharide.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. Adding papermaking sludge as an inorganic waste to the pullulan-producing culture medium can significantly enhance the pullulan-producing ability of *Bacillus buddingus* while maintaining its original viscosity level.

[0017] 2. It pioneered research on the correlation between papermaking sludge and pullulan, enriching the study on the reduction and resource utilization of papermaking sludge.

[0018] 3. In addition to enhancing the pullulan production capacity of *Bacillus brevicorum*, this invention also has the function of screening for high pullulan-producing *Bacillus brevicorum*, further enriching the germplasm resources for producing functional polysaccharides. Attached Figure Description

[0019] Figure 1 The effect of papermaking sludge on pullulan production by *Bacillus buddingus*.

[0020] Figure 2 The effect of papermaking sludge on pullulan viscosity. Detailed Implementation

[0021] The following embodiments are further explanations and illustrations of the present invention, but not limitations thereof.

[0022] This invention applies to pullulan-producing budding short-stalk molds, which can be screened or purchased from a fungal bank. The following examples use pullulan-producing budding short-stalk mold Aureobasidium pullulans E1 as an example for illustration.

[0023] Aureobasidium pullulans E1 is disclosed in the NCBI with accession number OP804136. The applicant also possesses this strain and guarantees its release to the public for 20 years from the date of application.

[0024] Example 1: Enhancing the ability of papermaking bleaching mud to produce pullulan polysaccharides from *Bacillus buddingus*.

[0025] The budding short-stalked spore strain E1 was streaked onto YPD plates and incubated statically at 30°C for 2 days. Colonies from the plates were picked and placed in YPD liquid medium, and cultured at 30°C and 220 rpm for 1.5 days to obtain a seed culture. Then, the seed culture was inoculated at an inoculum volume of 8% into pullulan-producing medium supplemented with papermaking sludge (0.1–0.5% w / vg / ml), and fermented at 30°C and 200 rpm for 4 days; the control group was fermented in the original medium without papermaking sludge. The fermentation broth was centrifuged at 12,000 rpm for 10 min to obtain the supernatant and bacterial cells. Polysaccharide yield and biomass were obtained by alcohol precipitation and differential gravimetric analysis, and the viscosity of the fermentation supernatant was measured using an NDJ-5S rotating digital viscometer.

[0026] like Figure 1 As shown (0% is the control group), with the increase of the amount of papermaking sludge added, the yield of pullulan in the fermentation process of *Brachystomata* strain E1 (4 days) first increased slowly and then decreased. The highest cumulative amount of pullulan (56.8 g / L) was achieved when the amount of papermaking sludge was 0.4% (w / v). Compared with the control group (0.379 g / g), the yield and efficiency of pullulan in the 0.4% (w / v) experimental group (0.473 g / g) increased by 24.8%, while the biomass did not change significantly throughout the fermentation process. This indicates that papermaking sludge increased the yield of pullulan without affecting cell growth. This may be because papermaking sludge can buffer the acidification process of the fermentation medium and provide trace elements such as potassium, magnesium, and iron, thereby promoting the enzyme activity of pullulan synthesis-related enzymes, increasing cell membrane permeability, and ultimately efficiently converting the substrate into pullulan. This indicates that adding papermaking sludge can enhance the ability of the budding short-stalked mold strain E1 to synthesize pullulan polysaccharide.

[0027] YPD liquid culture medium: by mass fraction, it includes 1% yeast extract, 2% peptone and 2% glucose, with the remainder being water; for solid plates, 1.5% agar by mass fraction needs to be added. The preparation method is to mix all components evenly and sterilize at 121°C for 20 minutes.

[0028] Original culture medium: by mass fraction, sucrose 12%, yeast powder 0.25%, K2HPO4 0.4%, NaCl 0.05%, (NH4)2SO4 0.04%, with the remainder being water. The preparation method is to mix all the components evenly and sterilize at 121℃ for 20 min.

[0029] The culture medium for producing pullulan in the experimental group consisted of the following components by mass fraction: 12% sucrose, 0.25% yeast extract, 0.4% K2HPO4, 0.05% NaCl, 0.04% (NH4)2SO4, and papermaking mud (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, w / vg / ml), with the remainder being water. The preparation method involved mixing all components thoroughly and sterilizing at 121℃ for 20 minutes.

[0030] Example 2: Effect of papermaking sludge on the viscosity of pullulan synthesized by *Bacillus brevis*

[0031] The viscosity of the fermentation supernatant obtained in Example 1 was measured. Figure 2 The results showed that (0% was the control group), compared with the control group, the viscosity of pullulan first increased and then decreased with the concentration of papermaking sludge (0.1–0.5%, w / v), reaching its highest viscosity at a concentration of 0.1% (w / v). The results of the other experimental groups were similar to those of the control group. Figure 1 The results indicate that papermaking sludge can provide high-viscosity pullulan, and while promoting increased pullulan yield and productivity, it maintains the viscosity of the polysaccharide without significant change. Normally, the viscosity of pullulan synthesized by *Bacillus buddingus* decreases as the yield increases. However, papermaking sludge significantly increased the pullulan production of *Bacillus buddingus* E1 strain while maintaining no significant change in viscosity. This may be because the 0.4% (w / v) concentration of papermaking sludge buffers the acidification process of the culture environment, and its inorganic nutrients accelerate the efficient expression of key enzymes in pullulan synthesis by *Bacillus buddingus*, including glucosyltransferase, UDPG pyrophosphorylase, and α-glucan synthase-2, enhancing cell membrane permeability. This allows papermaking sludge to simultaneously increase both the yield and viscosity of pullulan synthesized by *Bacillus buddingus* strain E1.

[0032] In conclusion, papermaking sludge, as an inorganic waste, can significantly enhance the ability of *Brachystomata buddingis* to produce pullulan while maintaining the original viscosity level of pullulan.

[0033] The above embodiments are merely illustrative examples of the present invention, and the scope of protection of the present invention is not limited thereto. Any modifications made by those skilled in the art within the scope of the technology disclosed in this invention, based on the spirit and solutions of this patent, are within the scope of protection of this invention.

Claims

1. A method for enhancing the ability of a paper white mud- reinforced Aureobasidium pullulans to produce pullulan, characterized by, Papermaking white mud is added to a culture medium for producing pullulan as inorganic waste, and Aureobasidium pullulans is used for fermentation to produce pullulan, wherein the inorganic waste papermaking white mud is directly added to the culture medium, and the specific steps are as follows: 1) activating the bacteria: the Aureobasidium pullulans strain is streaked on a YPD plate, and incubated at 25-35°C for 2-3 days to obtain the bacteria; 2) obtaining the seed liquid: the bacteria are picked and placed in a YPD liquid culture medium, and incubated at 25-35°C and 180-250 rpm for 1-2 days; 3) fermentation to produce sugar: the seed liquid is inoculated into a pullulan production culture medium containing papermaking white mud at a volume ratio of 5-10%, and incubated at 25-35°C and 180-250 rpm for 3-5 days; 4) detecting the polysaccharide and biomass: the fermentation liquid is centrifuged at 12000 rpm for 10 min, and the supernatant is alcohol precipitated and dried with the bacteria to obtain the polysaccharide yield and biomass; the viscosity of the supernatant is detected; the pullulan production culture medium containing papermaking white mud comprises, by mass fraction, 12% sucrose, 0.25% yeast powder, 0.4% K2HPO4, 0.05% NaCl, 0.04% (NH4)2SO4, and 0.4% papermaking white mud w / v; The Aureobasidium of the present application is Aureobasidium pullulans E1.

Images