CN102766656A

CN102766656A - Method for cheaply preparing microbial flocculant by utilizing bagasse

Info

Publication number: CN102766656A
Application number: CN2012102269117A
Authority: CN
Inventors: 尹华; 秦华明; 何宝燕; 叶锦韶; 彭辉; 张娜
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2012-07-03
Filing date: 2012-07-03
Publication date: 2012-11-07

Abstract

The invention provides a method for cheaply preparing microbial flocculant by utilizing bagasse. In the method, bagasse is utilized as only carbon source to prepare cheap culture medium, and high-yield cellulase-producing bacteria and microbial flocculant-producing bacteria are selected to realize the high yield of cellulase and the microbial flocculant by means of a fermentation process coupling the two strains of bacteria, and therefore the approach to bagasse resource utilization is broadened. Compared with the method which produces the microbial flocculant by directly utilizing cellulase to saccharify bagasse, the method dispenses with the cellulase purification process, and also effectively utilizes residual substrate left over after trichoderma fermentation to produce the flocculant, thus increasing the utilization rate of the material and reducing the discharge of wastewater as well, and the produced enzyme and flocculant are biodegradable and environment-friendly, and therefore secondary pollution cannot be generated. The fermentation method is simple and easy to control, and is favorable for mass production.

Description

A kind of cheap method for preparing microbial flocculant of bagasse of utilizing

Technical field

The present invention relates to the application technology as the second resource field of bagasse, be specifically related to a kind of cheap method for preparing microbial flocculant of bagasse of utilizing.

Background technology

Sugarcane is one of the most wide farm crop of whole world cultivated area, is mainly used in to refine sugar and system wine, and its back of squeezing the juice produces a large amount of bagasse, it is estimated that global every year, about 200,000,000 tons of bagasse need be handled.Bagasse is a kind of biomass that contain high-carbon high energy, mainly is made up of with other material 20% ~ 30% xylogen, 40% ~ 45% Mierocrystalline cellulose and 30% ~ 35% semicellulose and a spot of ash content.Compare with agricultural wastes such as stalk, semicellulose in the bagasse and Mierocrystalline cellulose be the high polymer for being made up of monose such as wood sugar, glucose all, makes bagasse more have exploitation to be worth.The bagasse utilization ratio is not high at present, and except that part was used to chopping and returning, fermentation producing methane, burning electricity generation, production ethanol, all the other major parts then were dropped, and have caused the waste of resource, the pollution of environment and the increase of processing pressure thereof.The higher value application degree that how to improve this biomass waste of bagasse has become the emphasis that countries in the world are paid close attention to.

Flocculation agent is widely used in the industries such as feedwater, wastewater treatment, foodstuff production and fermentation, is one type of maximum medicament of consumption in the processes such as process water, domestic water, wastewater treatment, dehydrating municipal sludge.But the preparation of traditional flocculant need consume a large amount of physical resources; In use usually can produce secondary pollution; Utilize microbial technique to prepare flocculation agent and then can realize changing rejected material to useful resource; Harmless to production unit and Pollutant Treatment facility, to the environment non-secondary pollution, human health and environment protection are had important practical significance.Though it is at present many to the research of microbial flocculant; But the product that can be applicable to actual production seldom; Its major cause is higher for cultivating cost; Therefore utilize waste to do and substitute substratum production flocculation agent, reducing its production cost is the important channel that promotes the microbial flocculant commercial application.

Production cost is the key factor of microbial flocculant practical application, and adopting waste is the effective way that reduces production costs as substituting the culture medium culturing bacterium for producing flocculant of microbe.At present; The alternative substratum that correlative study is both at home and abroad adopted is main with more rich liquid of organism such as carbon containing, nitrogen, phosphorus mainly, like the extracting solution of Dairy Wastewater, molasses containing waste water, starch wastewater, beer waste water, soy sauce waste liquid, tofu wastewater, gourmet powder waste water, sewage, fish meal wastewater, percolate, flower, hydrogen manufacturing black liquor etc.But all there are some problems in these cheap preparation materials, and are bigger like the composition and the concentration change of food processing wastewater, wherein the soy sauce production waste water COD of different batches _CrConcentration differs and can reach 100,000 mg/L, causes existing research not find propagable culture medium prescription and preparation technology as yet.Part food processing wastewater colourity is high, nutritive ingredient is unbalanced, contain the fermentating metabolism product that suppresses the growth of high-effective microorganism bacterium for producing flocculant, thereby causes microbial flocculant output lower.Therefore, seeking cultivated material cheap and easy to get, as to be prone to industrialization is even more important.

Semicellulose in the bagasse and Mierocrystalline cellulose be the high polymer for being made up of monose such as glucose, wood sugar, seminose and pectinoses all, can make glucose small molecules and other monose from bagasse, be released out after handling through methods such as physics, chemistry, biologies.Physical method mainly is to pulverize, microwave treatment and steam explosion, and cost is high, and productive rate is low.Chemical process is divided into the hydrolyzation catalysis effect of acid or enzyme.The acid system hydrolysis generally needs acidproof, heat-resisting, withstand voltage specific installation, is prone to produce by product, influences follow-up fermentation.Enzymatic hydrolysis has advantages such as equipment is simple, and reaction conditions is gentle, and raw material saccharification yield is high, and by product is few and free from environmental pollution, but used cellulase price is high.And utilize can cellulolytic one type of bacterium (, aspergillus mould and mould etc.) fermentation like wood, be the direction of saccharification of cellulose technological development.This method has the reaction conditions gentleness, does not produce pollution substance, is easy to control.Disadvantages affect such as but reaction time is long, efficient is low the widespread use of microbiological treatment cellulose materials.

The present invention directly is matrix with bagasse; Realized the coupling that bagasse saccharification and microbial flocculant are produced through asynchronous fermentation; For the production cost that reduces microbial flocculant provides technical foundation, this will be significant with the recycling approach of expanding agricultural wastes to the commercial application of microbial flocculant.

Summary of the invention

The objective of the invention is to overcome the above-mentioned deficiency that prior art exists, a kind of cheap method for preparing microbial flocculant of bagasse of utilizing is provided.The present invention chooses the cellulase producing bacteria and the bacterium for producing flocculant of microbe of high yield, through the zymotechnique of the two strain bacterium that are coupled, makes full use of the cheap substratum of bagasse preparation, and fermenting process is simple, controls easily, can reduce the production cost of flocculation agent greatly.The object of the invention is realized through following technical scheme.

A kind of cheap method for preparing microbial flocculant of bagasse of utilizing comprises the steps:

(1) preparing culture medium: the carbon source consumption is 10 ~ 50g/L in the substratum of preparation, and the nitrogenous source consumption is 1 ~ 5g/L, KH ₂PO ₄Consumption 1 ~ 5 g/L, MgSO ₄Consumption 0.1 ~ 0.5 g/L, all the other are tap water; Said carbon source is a bagasse;

(2) fermentation: in fermentor tank, add the substratum of being prepared in the step (1); Utilize HP steam heating tank body; Making tank pressure maintain between 0.1 ~ 0.11MPa sterilizes; Utilize water coolant to make fermentor tank be cooled to 23 ~ 35 ℃ of leavening temperatures with stirring to cool off, the adjusting stir speed (S.S.) is 100 ~ 300rminP ^-1(preferred 130 rminP ^-1), the control air flow is 80 ~ 150mLminP ^-1P(preferred 100 mLminP ^-1) and keep tank pressure 0.015 ~ 0.025 MPa (preferred 0.02MPa) after, insert the mould spore liquid of fresh wood that accounts for culture volume 1% ~ 5%, the absorbance A of spore liquid=0.6, inoculation culture 24 ~ 48h; Fermentation time is very crucial in this fermenting process, and the short living weight of incubation time is low, and bagasse saccharification efficient is not high; The secretory product that the long wood of incubation time is mould and the hydrolysate of substrate make the complicated component of fermented liquid, and some factor can influence follow-up production by biological flocculation agent.Therefore, the wooden mould fermentation condition and the working condition of flocculation agent will be coupled.

(3) flocculation agent production: step (2) fermented liquid is adjusted to pH 4 ~ 6, and the sterilization postcooling is to room temperature, and regulating stir speed (S.S.) is 100～300 rminP ^-1P(preferred 130rminP ^-1P), the control air flow is 80 ~ 150mLminP ^-1P(preferred 100 mLminP ^-1) and keep tank pressure 0.015 ~ 0.025 MPa (preferred 0.02MPa) after, With 0.05 ~ 0.25g/L inoculum size25 ~ 37 ℃ of microbe inoculation bacterium for producing flocculant leavening temperatures, fermentation time 36 ~ 72h; Collect nutrient solution, thalline is removed by filter, obtain having the fermented liquid of flocculation activity, measure its flocculation activity with the Kaolin clay suspension of 400NTU with gauze.

Further optimize, also comprise the flocculation activity of measuring fermented liquid with the Kaolin clay suspension of 400NTU in step (3) with flocculation activity.

Further optimize, the inoculation of bacterium for producing flocculant of microbe is a mycelium inoculation in the step (3).

Further optimize, all adopting the bottom aeration to add the saturation dissolved oxygen that stirs the control dissolved oxygen in step (2) and the step (3) in the fermenting process is 30 ~ 80%.

Further optimize, the said carbon source of step (1) is the bagasse after pulverizing.Said nitrogenous source is NaNO ₃, (NH ₄) SO ₄, NH ₄NO ₃, Ca (NO ₃) ₂And urea.

Further optimize, the pH value of the said substratum of step (1) is 3 ~ 7.

In the above-mentioned substratum, selected nitrogenous source low price, and consumption is few; The preparation of use tap water can replenish some micro-factors of microorganism growth and cheaply be easy to get than other pure water.In a word, above-mentioned culture medium prescription is simple, and selected all is raw materials cheap and easy to get, greatly reduces the cost of microorganism culturing, is suitable for large-scale industrial production.

Further optimize, in the step (2), the liquid volume added of substratum is the half the of fermentor tank volume, and said HP steam is the 0.4MPa HP steam, and said sterilization is that the tank body temperature maintenance is at 120 ℃ of sterilization 30min; Sterilization described in the step (3) is 110 ℃ of sterilization 20min, and said bacterium for producing flocculant of microbe consumption is in weight in wet base, and water ratio is 87%.

Further optimize, the said wood of step (2) is mould be Trichodermareesei ( Trichoderma reesei), koning trichoderma ( Trichoderma Koningii), viride ( Trichoderma viride) in a kind of.

Further optimize, the flocculating rate that step (3) obtains having the fermented liquid of flocculation activity is 80～90%, and flocculation agent content is 1.05 ~ 1.40g/L.

The above-mentioned bagasse cheapness of utilizing prepares in the method for microbial flocculant, and flocculation agent determination of yield method is: get the fermented liquid of 25mL 4 ℃ of absolute ethyl alcohols of volume ratio adding with 1:2, and after 4 ℃ of lucifuges leave standstill 24h, 4 ℃ of following 6000rminP ^-1P, centrifugal 10min, to throw out with the washing with alcohol of same concentrations 2 times after, dry to constant weight for 60 ℃, measure its quality.

The present invention compares with prior art, has following advantage and effect:

(1) fermention medium of the present invention's employing as sole carbon source, has only added a small amount of nitrogenous source and trace element with bagasse, greatly reduces the production cost of flocculation agent.

(2) technology that the present invention adopted has realized the coupling of bagasse Mashing process and microbial flocculant production technique, and fermentation time is short, greatly reduces power consumption, has practiced thrift production cost.Produce microbial flocculant with DIRECT UTILIZATION OF CELLULOSE enzyme glycolysis bagasse and compare, both saved the purification process of cellulase, the while has effectively been utilized the residue substrate produce flocculant after the wooden mould fermentation, has improved utilization ratio of raw materials and has also reduced discharge of wastewater.

(3) fermentation process of the present invention is easy, and control helps large-scale production easily.

(4) enzyme that the present invention produced and flocculation agent have biodegradability and environment friendly, can not produce secondary pollution.

(5) the present invention has realized the high yield of cellulase and microbial flocculant simultaneously, has expanded the approach of bagasse recycling.

Embodiment

Below in conjunction with embodiment the present invention is further described, but embodiment of the present invention and protection domain are not limited thereto.

Embodiment 1

Preparation fermention medium concentration is 20g/L bagasse, 1.6g/L (NH ₄) ₂SO ₄, 2g/L KH ₂PO ₄, 0.1 g/L MgSO ₄, sterilization back pH6.0 inoculates 1% viride spore liquid, cultivates 48h down for 30 ℃.Fermented liquid is adjusted to 110 ℃ of sterilization 20min behind the pH5.5, is cooled to room temperature then, inoculation Aspergillus sojae mycelia (inoculum size 0.05g/L), the saturation ratio of dissolved oxygen maintains 32 ~ 51%, 30 ℃ of leavening temperatures, fermentation time 50h.Collect nutrient solution, with gauze elimination thalline, the fermented liquid that obtains having flocculation activity is got this fermented liquid of 1ml and is added to 100ml kaolin suspension (400NTU) to measure its flocculating rate be 83%.Other gets its flocculation agent content of 25ml fermented liquid mensuration is 1.25g/L.

Embodiment 2

Preparation fermention medium concentration is 30g/L bagasse, 2.0g/L (NH ₄) ₂SO ₄, 2.5g/L KH ₂PO ₄, 0.1 g/L MgSO ₄, sterilization back pH6.0 inoculates 1% viride spore liquid, cultivates 40h down for 30 ℃.Fermented liquid is adjusted to 110 ℃ of sterilization 20min behind the pH6, is cooled to room temperature then, inoculation Aspergillus sojae mycelia (inoculum size 0.10g/L), the saturation ratio of dissolved oxygen maintains 35 ~ 48%, 32 ℃ of leavening temperatures, fermentation time 50h.Collect nutrient solution, with gauze elimination thalline, the fermented liquid that obtains having flocculation activity is got this fermented liquid of 1ml and is added to 100ml kaolin suspension (400NTU) to measure its flocculating rate be 85%.Other gets its flocculation agent content of 25ml fermented liquid mensuration is 1.30g/L.

Embodiment 3

Preparation fermention medium concentration is 25g/L bagasse, 1.8g/L (NH ₄) ₂SO ₄, 2.1g/L KH ₂PO ₄, 0.2 g/L MgSO ₄, sterilization back pH6.0 inoculates 1% viride spore liquid, cultivates 48h down for 32 ℃.Fermented liquid is adjusted to 110 ℃ of sterilization 20min behind the pH5, is cooled to room temperature then, inoculation Aspergillus sojae mycelia (inoculum size 0.15g/L), the saturation ratio of dissolved oxygen maintains 30 ~ 46%, 35 ℃ of leavening temperatures, fermentation time 48h.Collect nutrient solution, with gauze elimination thalline, the fermented liquid that obtains having flocculation activity is got this fermented liquid of 1ml and is added to 100ml kaolin suspension (400NTU) to measure its flocculating rate be 86%.Other gets its flocculation agent content of 25ml fermented liquid mensuration is 1.35g/L.

Embodiment 4

Preparation fermention medium concentration is 20g/L bagasse, 1.6g/L (NH ₄) ₂SO ₄, 2g/L KH ₂PO ₄, 0.1 g/L MgSO ₄, sterilization back pH6.0 inoculates 1% viride spore liquid, cultivates 48h down for 30 ℃.The filtering fermentation liquor that obtains is removed thalline, and the centrifugal 10min of 4000rpm, the fermented liquid of obtaining record CMC enzyme value alive and be 1.35U, and reducing sugar content is 0.26g/L.

Claims

1. a method utilizing bagasse to prepare microbial flocculants cheaply, is characterized in that comprising the steps:

(1) Preparation of medium: the amount of carbon source in the prepared medium is 10~50g/L, the amount of nitrogen source is 1~5g/L, the amount of KH ₂ PO ₄ is 1~5 g/L, and the amount of MgSO ₄ is 0.1~0.5 g/L, the rest is tap water; the carbon source is bagasse;

(2) Fermentation: Add the culture medium prepared in step (1) into the fermenter, heat the tank with high-pressure steam to maintain the pressure of the tank at 0.1-0.11MPa for sterilization, and use cooling water and stirring to cool the tank. Cool the fermenter to a fermentation temperature of 23~35°C, adjust the stirring rate to 100~300r·minP ^-1 , control the air flow to 80~150mL·minP ^-1P and maintain the tank pressure at 0.015~0.025 MPa, then insert the medium volume 1%~5% fresh Trichoderma spore liquid, the absorbance of the spore liquid is A=0.6, inoculate and culture for 24~48h;

(3) Flocculant production: adjust the fermentation broth in step (2) to pH 4~6, cool to room temperature after sterilization, adjust the stirring rate to 100~300 r·minP ^-1P , and control the air flow to 80~150mL·minP ^-1P and maintain the tank pressure at 0.015~0.025 MPa, inoculate microbial flocculant producing bacteria with 0.05~0.25g/L inoculum. Fermentation temperature is 25~37℃, fermentation time is 36~72h; collect the culture medium and filter the bacteria with gauze removed to obtain a fermented broth with flocculation activity.

2. A method for preparing microbial flocculants cheaply by using bagasse according to claim 1, characterized in that step (3) further comprises measuring the flocculation activity of the fermented liquid with flocculation activity by using 400 NTU kaolin suspension.

3. A method for cheaply preparing microbial flocculants by using bagasse according to claim 1, characterized in that the inoculation of microbial flocculant-producing bacteria in step (3) is mycelia inoculation.

4. A method for preparing microbial flocculants cheaply using bagasse according to claim 1, characterized in that bottom aeration and stirring are used to control dissolved oxygen saturation in the fermentation process in step (2) and step (3) 30~80%.

5 . The method for preparing microbial flocculants cheaply by using bagasse according to claim 1 , characterized in that the carbon source in step (1) is crushed bagasse. 6 .

6. A method for cheaply preparing microbial flocculants using bagasse according to claim 1, characterized in that the nitrogen source in step (1) is NaNO ₃ , (NH ₄ )SO ₄ , NH ₄ NO ₃ , Ca (NO ₃ ) ₂ and urea.

7. A method for cheaply preparing microbial flocculants by using bagasse according to claim 1, characterized in that the pH value of the culture medium in step (1) is 3-7.

8. A method for cheaply preparing microbial flocculants using bagasse according to claim 1, characterized in that in step (2), the amount of medium added is half of the volume of the fermenter, and the high-pressure steam is 0.4 MPa high-pressure steam, the sterilization is to maintain the temperature of the tank at 120°C for 30 minutes; the sterilization in step (3) is to sterilize at 110°C for 20 minutes, and the amount of microbial flocculant-producing bacteria is calculated by wet weight, containing water The rate is 87%.

9. A method for preparing microbial flocculants cheaply using bagasse according to any one of claims 1 to 8, characterized in that the Trichoderma described in step (2) is Trichoderma reesei, Trichoderma konshii, green A type of Trichoderma.

10. A method for cheaply preparing microbial flocculants using bagasse according to claim 9, characterized in that the flocculation rate of the fermented liquid with flocculation activity obtained in step (3) is 80-90%, and the flocculant content is 1.05 ~1.40g/L.