CN105907806A - Method for preparing polyhydroxyalkanoate from poisonous industrial pollutant phenol - Google Patents
Method for preparing polyhydroxyalkanoate from poisonous industrial pollutant phenol Download PDFInfo
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Abstract
The invention belongs to the technical field of environmental pollution and resource comprehensive utilization, and particularly relates to a method for preparing polyhydroxyalkanoate from a poisonous industrial pollutant phenol. The method comprises the steps that excess activated sludge of an urban sewage treatment plant is used as inoculum, the activated sludge is acclimatized to completely adapt to high phenol concentration, and the phenol with the high concentration can be completely degraded within a short time; the concentrated sludge is mixed with a nutrient solution containing nitrogen, phosphorus, calcium, magnesium, iron and the like to form a reaction mixed solution; phenol is added in, and cell growth and PHA accumulation start to be carried out. Various operation conditions are explored, the influences of the operation conditions on sludge growth and intracellular PHA accumulation are measured, and therefore an optimal parameter range is determined, wherein the operation conditions comprise the carbon nitrogen ratio in growth media, the medium pH value and dissolved oxygen level during phenol degradation, the calcium, magnesium and iron concentration, salinity, temperature and F/M ratio in the growth media, and the phenol adding way.
Description
Technical field
The invention belongs to environmental protection and technical field of resource comprehensive utilization, particularly relate to the method that toxic organic pollutant in industrial wastewater prepares polyhydroxyalkanoate.
Background technology
Polyhydroxyalkanoate (polyhydroxyalkanoates is called for short PHA) is a kind of intracellular polymers of the macromolecule synthesized in vivo by microorganism.Its basic structure is the fatty acid mono containing hydroxyl, forms ester bond, form linear polymeric further between the hydroxyl of two adjacent monomers.PHA is similar to plastics in nature, and has various of monomer structure and various material behavior.Because its production process is natural biological respinse, this type of material also has good bio-compatibility and degradability.Owing to it has broad application prospects at aspects such as Packaging Engineering, organizational project, slow-release material and medical materials, the biggest research and application interest are attracted in recent years.
In general, the premise of intracellular synthesis PHA is that microorganism is in undulatory property and unbalanced growing environment.Modal situation include in somatomedin carbon source too much, the element such as nitrogen/phosphorus/sulfur very few, or food/microorganism ratio (food to microbe ratio, it is called for short F/M ratio) too high, the cell proliferation rate thereby resulted in is restricted, it is impossible to be used for growing by too much carbon source in time.These carbon sources can be converted into PHA and be stored in cell interior, for further growth during improvement of terms after remaining by now microorganism.Additionally, the too low substrate caused of dissolved oxygen levels cannot Quick Oxidation, and aerobic/anaerobic condition alternately can also promote the accumulation of PHA.It is sequencing batch type culture process that another kind is considered the operating condition of beneficially PHA synthesis, the most in batches, intermittently adds carbon source, makes microorganism experience/hunger period (feast/famine) of being satiated with food repeatedly.Now have some microorganisms to select too much or that growth can not be used at once carbon source to be converted into PHA, as adapting to the unstable means with acute variation growing environment, balance its value-added speed, in microbial population, increase competitive advantage for it.
PHA can be synthesized as substrate by various different organic substances.Up to the present, most of research and commercial applications use pure nontoxic simple organic, and if acetate, glucose etc. are as carbon source, purebred bacterium carries out PHA production as instrument.Its research emphasis first is bacterial screening, purification, genetic modification (genetic engineering bacterium), and second is operating procedure and the development of equipment and optimization, to improve the overall PHA production efficiency of the PHA ratio sum in biomass.But use purebred bacterium and pure material to have it to limit to as material.This type of carbon source itself relatively costly, and keep the purity of flora to have higher requirement also for operating procedure in process of production.The most in recent years in order to reduce cost, several researchers have proposed use mixing city/industrial or agricultural effluent sewage as carbon source, in purebred or mixed bacterial, synthesize PHA.The effluent sewage having obtained research at present includes city domestic sewage, activated sludge fermentation liquid, food and sugar industry wastewater, printing and dyeing and paper waste and agricultural biomass garbage etc..But these substrates have a common ground, the most nontoxic, the organic substance of toxic effect for microorganism is used to lack very much as the research of substrate.It is raw material that Maskow et al. [1] utilized phenol in 2000, cultivate as operational means continuously, has successfully synthesized PHA in two kinds of purebred bacterium.And Reddy et al. [2] recently tests multiple aldehydes matter, find that they all can be as the substrate of purebred bacterium synthesis PHA.The inventor team of this patent further study under sequencing batch type operating condition, phenol is as the PHA accumulation situation in mixed bacterial during sole carbon source, find its can reach more than 50% dry cell weight [3], and the situation of some purebred bacterium+non-toxic substrate is close.This is to studies have reported that mixed bacterial utilizes toxic substrate to carry out PHA synthesis for the first time, and its result shows good application prospect.Therefore the present invention deepens research further, it is instrument using phenol as representative, the mixed bacterial activated sludge tamed completely of toxic industrial pollutant, in batches/sequencing batch type is cultivated as operating procedure, deeply and carefully studies and optimizes the synthesis condition of PHA, form the most set of technical scheme.
List of references:
[1] Maskow, T. Babel W. Calorimetrically recognized maximum
yield of poly-3-hydroxybutyrate (PHB) continuously synthesized from toxic
substrates. Journal of Biotechnology 2000, 77, 247-253.
[2] Venkateswar Reddy, M.; Mawatari,
Y.; Yajima, Y.; Seki, C.; Hoshino T.; Chang, Y.-C.
Poly-3-hydroxybutyrate (PHB) production from alkylphenols,
mono and poly-aromatic hydrocarbons using
Bacillus sp. CYR1: A new strategy for wealth from waste. Bioresource Technology 2015, 192, 711-717.
[3] Wosman, A.; Lu, Y.H.; Sun, S.P.; Liu, X.; Wan, C.L.; Zhang,
Y.; Lee, D.-L.J.; Tay. J.H. Effect of operational
strategies on activated sludge’s acclimation to phenol, subsequent aerobic
granulation, and accumulation of polyhydoxyalkanoates.
Journal of Hazardous Materials, 2016 , 317, 221-228.。
Summary of the invention
It is an object of the invention to provide a kind of method utilizing toxic organic pollutant in industrial wastewater to prepare polyhydroxyalkanoate.
The method preparing polyhydroxyalkanoate that the present invention provides, concretely comprises the following steps:
(1) use the residual active sludge of municipal sewage plant as inoculum.To this activated sludge through certain domestication process [3], this mud is made to adapt to high phenol concentration (such as more than 500mg/ L) completely, can be degradable complete (such as 30-60min) at short notice by the phenol of this concentration；
(2) by the mixed bacteria mud adapting to high phenol concentration completely obtained above, cultivating in being placed on reactor, every two to three days adds the nutrient substance such as phenol, nitrogen, phosphorus, calcium, magnesium, a ferrum, maintains and cultivates；
(3), when carrying out PHA production, a mixed bacteria mud taking-up part is carried out high speed centrifugation separation every time, precipitation, obtain thickened sludge；
(4) this thickened sludge is mixed with nutritional solutions such as nitrogen, phosphorus, calcium, magnesium, ferrum, and add a certain amount of alkaline buffer solution (with NaHCO3Form), form the reaction mixture of a batch；
Wherein, the concentration of these nutrient substance can be adjusted respectively, forms certain Concentraton gradient (such as the magnesium elements etc. of 0,2.5,5 mg/L)；Adjust the initial original thickened sludge taken out, make this reaction mixture have certain initial sludge concentration, such as 0.5,1.0,2.0 g/L etc..This reaction mixture is carried out aeration aerating and stirring mixing, can dissolved oxygen concentration in change system by the intensity of regulation aeration；
(5) it is subsequently adding a certain amount of phenol so that it is there is certain initial concentration (such as 250,500,1000,2000 mg/L).Owing to phenol is unique organic carbon source in body series, the concentration of phenol also can calculate initial carbon concentration (concentration of carbon=76.6% phenol concentration).This concentration then be can get compared with the nitrogen concentration of element of addition initial carbon weight nitroxide ratio (C/N,
W/w).Initial ratio between phenol concentration and sludge concentration is then the F/M ratio (such as 0.1,0.4,0.8 etc.) of this batch.After phenol adds, mud i.e. starts cell growth and PHA accumulation (can monitor and compare both speed in the process, determine the effect of each factor).Until this phenol added is degradable complete, process as a phenol and the batch of PHA accumulation；
(6) after the phenol degrading in a upper batch terminates, again add the nutrient such as a certain amount of phenol and nitrogen, phosphorus, phosphorus, calcium, magnesium, ferrum, start next batch, to continue to improve the total amount of PHA and the ratio in intracellular.To with a collection of mud adds this mode of phenol repeatedly, it is sequencing batch type and processes (sequencing
Batch mode).
In the present invention, nitrogen element can be with NH4Cl form, P elements can be with K2HPO4Form, calcium constituent can be with CaCl2Form, magnesium elements can be with MgSO4Form, ferrum element can be with FeSO4Form adds.
In the present invention, course of reaction also can add NaCl to change the salinity of system.
In the present invention, operate under different ambient temperatures, the temperature impact for PHA accumulation can be tested.
Control in this process, change various operating condition, measure the impact that they accumulate for mud growth and intracellular PHA, to determine the parameter area more optimized.These operating conditions include: the pH value in medium and calcium, magnesium, concentration of iron, salinity, temperature, F/M ratio and phenol dosing method in dissolved oxygen levels, somatomedin when carbon-nitrogen ratio in somatomedin, phenol degrading.Wherein, carbon-nitrogen ratio is in the range of 5-40(w/w)；0.3-4.3 mg/L, 0-7.2 mg/L, 0-4.8 mg/L, 0-9 mg/L it is respectively in the range of phosphorus, calcium, magnesium, concentration of iron；Preferably it is respectively 0.3-4.3 mg/L, 1-7.2 mg/L, 0.5-4.8 mg/L, 1-9 mg/L in the range of phosphorus, calcium, magnesium, concentration of iron；Dissolved oxygen concentration is in the range of 1 mg/L-saturated concentration (about 8 mg/L)；The buffer solution amount added by regulation and the pH value of reaction is controlled between 4-8.5；Salinity range is that 0%-4%(is in terms of NaCl)；Temperature range is 7-23 ° of C.Initial phenol concentration scope is 250-2000 mg/L；Initial sludge concentration scope is 0.5-2.5 g/L；F/M is 0.1-1.6 than scope；Phenol dosing method then can be divided into and disposably adding (single batch processed) and the most repeatedly add (sequencing batch type process).
Result shows, carbon-nitrogen ratio is at 5-40(w/w) between, dissolved oxygen levels is at 4-8
Time between mg/L, mud growth and intracellular PHA accumulation all can reach higher speed, and when same element phosphor (0.3-4.3 mg/L), calcium (0-7.2 mg/L), magnesium (0-4.8 mg/L), ferrum (0-9 mg/L) are in these concentration ranges, the speed of cell growth and PHA accumulation is the highest.It is thus determined that these concentration ranges are the operating conditions more optimized, higher PHA accumulation efficiency when the respective element in actual waste water changes in the range of these, all can be obtained.LDO level (< 0.5 mg/L) hinder growth and PHA to synthesize, optimal dissolution oxygen level is then established between 1-4 mg/L, and now PHA synthesis is very fast and aeration energy consumption is less.In somatomedin, add basic buffering substance control ph, find that when pH drops to less than 5, growth of microorganism and PHA synthesis all tend to stagnating.And adding suitable buffer substance, and pH value being controlled between 6.5-8.5, then both all can be normally carried out and without significant difference.Use three phenol concentration (250/500/1000 mg/L) and three initial sludge concentration (0.5/1.0/1.5 g/L) combinations, finding under low/medium/high three phenol concentration levels, improving initial sludge concentration for accelerating growth and PHA synthesis all has positive effect.Initial phenol concentration in somatomedin is the most relatively low, and the speed of growth and PHA accumulation speed are the highest, and high concentration has certain inhibitory action for the two process.For raising PHA is in the ratio of intracellular, under high sludge concentration, the difference of low/medium/high three phenol concentration is little；And under sludge medium concentration, the phenol of intermediate concentration has more preferable effect for high/low two levels.For the raising that total PHA measures, the combined efficiency of high phenol concentration+high sludge concentration is the highest.When industrialized production as the phenolic waste water (such as 1000-2000 mg/L) of high concentration need to be processed, sludge concentration should be brought up to 2 more than g/L, to alleviate inhibitory action and to obtain higher PHA production efficiency.
Test sequencing batch type technique further to imitate the situation of multiple batches of Continuous Wastewater Treatment in Practical Project.Use overall phenol dosage constant, the method that point once/secondary/tetra-time add variable concentrations phenol.Find disposably to add the operational approach of high concentration phenol and extend the time of whole processing procedure, but improve final PHA at the ratio of intracellular and the productivity of final PHA；And add Low Concentration Phenol in four times and add intermediate concentration phenol at twice, it processes, and the time is similar, and final PHA productivity and PHA are also closer in the ratio of intracellular, but both are all less than the situation disposably adding high concentration.If therefore needing to shorten overall processing time, then use the mode repeatedly adding Low Concentration Phenol the most efficient；And if target is the cell obtaining and having the highest PHA ratio, then can use the disposable mode adding high concentration phenol.This kind of mode the most more meets the process requirement of high-concentration industrial-water simultaneously, simplifies integrated artistic.
When sequencing batch type processes beginning, the PHA dry ratio (PHA%) of intracellular is inverse correlation with the speed of growth and PHA accumulation.When i.e. reaction starts, the intracellular PHA level having built up is the lowest, adds cell growth the fastest, PHA accumulation after phenol starts reaction the fastest.After intracellular PHA ratio reaches a higher level, accumulating rate is the most slack-off or even stagnates, and cell growth and phenol degrading also slow down.Finally, it was observed that the dry ratio of intracellular PHA has a higher limit, and under the experimental condition of the present invention, this value is in the range of 55-60%.It is thus determined that the most continuable PHA mode of production, i.e. after the PHA accumulation of a batch, taking out a part of mud as extracting the raw material of PHA, remaining mud then experiences a hunger period to reduce its intracellular PHA ratio, then as the material of beginning next batch.
Low temperature (7-13 DEG C) synthesizes for growth of microorganism and PHA obvious inhibitory action, but reaction still can be carried out.And speed is substantially accelerated, it is taken as that this is the most suitable temperature range at middle high temperature 15-23 DEG C.In the range of 0-2% salinity (in terms of NaCl), cell growth and PHA accumulation become negatively affected, and its speed is less than the condition of 0% salinity, but this impact is reversible, and the infringement that 4% salinity produces is the most irreversible.Therefore the impact of high salinity should be avoided in practical operation.
Accompanying drawing explanation
Fig. 1. carbon-nitrogen ratio is for cell growth and the impact of PHA accumulation.
Fig. 2. add various dose ealkaline buffer for cell growth and the impact (500 mg/L phenol, C/N=10) of PHA accumulation.
Fig. 3. in solution, dissolved oxygen levels is for cell growth and the impact (500 mg/L phenol, C/N=10) of PHA accumulation.
Fig. 4. the lower growth of different initial phenol/sludge concentration combination and the comparison (C/N=10) of PHA accumulation speed.
Fig. 5. different modes sequencing batch type adds phenol to growth and the impact of PHA accumulation.
Fig. 6. under similar initial sludge concentration, different initial intracellular PHA level is for growth and the impact of PHA accumulation.
Fig. 7. salt impact is for cell growth and the impact of PHA accumulation.
Detailed description of the invention
The present invention obtains residual active sludge from Shanghai one municipal sewage plant, carries out being directed to the domestication of high concentration phenol as inoculum.Use sequencing batch type technological operation reactor, step up the phenol concentration in reactor water inlet two middle of the month, finally give the mixed bacteria activated sludge adapting to 500 mg/L phenol concentration.Reactor mixed liquor is taken out, after high speed centrifugation separates, obtains mud, mix with the somatomedin of definite composition, and carry out aeration and stirring.This somatomedin comprises the components such as nitrogen, phosphorus, ferrum, calcium, magnesium, trace element and alkaline buffer, and these components can change concentration respectively to study the impact of its cell growth and PHA accumulation.The phenol of various dose is added under conditions of somatomedin and mud are thoroughly mixed, the index that periodically absorbance (OD600) under sampling monitoring 600nm grows as cell, adding acid Nile red dye dyeing PHA the most in the sample, the fluorescence intensity of monitoring product is as the index of PHA content.Repeated sampling is until the phenol added is degradable complete, as a reaction batch；Again add phenol and repeat above procedure, to simulate sequencing batch type reaction process.Determine that the impact of operating condition is as follows by above step test:
a. Carbon-nitrogen ratio (C/N) affects:At two initial sludge concentration (0.5/1.4
G/L) under and two initial phenol concentration (250/500 mg/L) respectively parallel test C/N ≈ 5,10,20,40(w/w) these levels.Result shows that the change in the range of this is the most notable for the impact of cell growth and intracellular PHA accumulation, all can reach to grow faster and PHA aggregate velocity.Therefore in practical operation, C/N in the range of this may be considered suitable condition.Under low mud/low phenol concentration, low ratio of carbon to ammonium (C/N=5) can slightly promote phenol degrading and cell growth, and C/N=40 slightly improves the content of final intracellular PHA.Data are shown in Fig. 1 (abscissa-response time/min).
b. pH Impact with interpolation buffer:Owing to phenol degrading produces organic acid, therefore the present invention uses interpolation alkaline buffer NaHCO3Method neutralize acidic materials, keep pH value.Parallel set up three samples, add 0 mL/5 mL/10 mL NaHCO respectively3 (0.5
Mol/L), to determine the impact for process of the pH value in degradation process.Finding in the case of without buffer, the pH value in solution quickly drops to about 4, and cell growth simultaneously and PHA tend to stagnating.And in the case of adding buffer, pH also decreases, and is reduced to about 6.5 when adding 5 mL, and when adding 10 mL, whole process pH is all higher than 7, illustrates that acidic materials are all neutralized.In the case of the two, cell growth and PHA are all not affected by significantly negatively affecting, and both growth rates and PHA accumulation speed are basically identical and higher.Thus the environment of the neutral meta-alkalescence of explanation is more beneficial for cell growth and PHA accumulation, and the pH of sour environment, especially less than 5 seriously hinders the two process.Data are shown in Fig. 2 (abscissa-response time/min).
c. The impact of dissolved oxygen levels:The method that the present invention uses fixing stir speed (S.S.) and changes aeration intensity, have studied growth and the situation of PHA accumulation under three dissolved oxygen levels (low/medium/saturated).Finding under medium dissolved oxygen levels 4-5 mg/L and high (saturated) dissolved oxygen levels about 10 mg/L, growth and PHA accumulation do not have the biggest difference and higher.Therefore in practical operation, the dissolved oxygen concentration of this scope is suitable condition.LDO horizontal 0.4-1 mg/L then slow down the two process, and after bringing up to 1-2 mg/L, PHA accumulation speed is significantly improved.Owing to aeration electric power to be consumed itself increases cost, therefore speculating in the conditions of the invention, PHA accumulation has an optimal dissolved oxygen levels at 1-4 mg/L, can reach higher speed and produce relatively low energy consumption.Concrete data are shown in Fig. 3 (abscissa-response time/min).
d. Phosphorus, calcium, magnesium, the impact of concentration of iron:Use without/intermediate concentration/twice intermediate concentration three concentration level, the effect of these elements of parallel study.Find cell growth and PHA accumulation no significant difference under three concentration ranges, it can be considered that the level change of these elements does not makes significant difference (the most another attached data) to (1-2 hour) growth of microorganism in a short time and intracellular PHA cumulative process.In practical operation, phosphorus 0-9 mg/L in the range of may be considered suitable condition at 0-7.2 mg/L, magnesium at 0-4.8 mg/L, ferrum at 0.3-4.3 mg/L, calcium.
e. F/M The impact of ratio:It is generally believed that the accumulation that higher F/M is than beneficially PHA, because too much carbon source can not be immediately available for growth by limited microorganism, thus it is converted into PHA and stores.But also have research to think, when F/M is relatively low, the contention for carbon source between microorganism can be aggravated in mixed bacterial, have some microorganisms now by strengthening PHA synthesis capability to fight for limited carbon source, thus keep the competitive advantage of oneself.Use this kind of toxic substrate of phenol time, situation is the most increasingly complex because phenol for microorganism toxic action with its in system concentration positive correlation, the highest microorganism concn then can alleviate murder by poisoning and the inhibitory action of phenol to a certain extent.
In order to preferably study the impact of F/M ratio, devise a test matrix.Use three initial phenol concentration and three initial sludge concentrations, study the growth under each combination condition and PHA accumulation situation respectively.These three phenol concentration is respectively 250,500,1000 mg/L, and three initial sludge concentrations are then 0.55-0.65,0.9-1.3,1.4-1.7
g/L.Observe in reality is tested, great majority reaction presents linear reaction kinetic characteristics, it is therefore assumed that growth and PHA synthesis are all Pseudo-zero order reaction (pseudo-zero order reaction), use the linear segment in growth and PHA concentration curve, carry out linear fit, try to achieve Pseudo-zero order reaction constant (pseudo-zero order rate constants).Listing these data in Fig. 4 to compare, wherein PHA ratio employs mark (rather than percent).
For growth rate, it is proportionate with initial sludge concentration, and is inverse correlation with initial phenol concentration.I.e. phenol concentration is the highest, and the growth rate under certain initial sludge concentration is the lowest, and this illustrates that phenol truly has inhibitory action for cell growth.The situation of PHA accumulation is the most complex, and under low sludge concentration, the total PHA concentration change trend in system is identical with cell growth tendency, and the lowest phenol concentration is more beneficial for this process.And under sludge medium concentration, the highest speed comes across (0.0138 g/L, min@500 mg/L) under medium phenol concentration, and the speed under 250/1000 mg/L is more or less the same (respectively 0.0099/0.0089 g/L, min).Under high sludge concentration, the highest speed then comes across 1000
Under mg/L phenol concentration (0.0251 g/L, min), and 250/500
Speed under mg/L is closer to (respectively 0.0202/0.0178 g/L, min).For PHA is in intracellular ratio, the trend of low/medium sludge concentration is similar to PHA total concentration, and under high sludge concentration, the highest rate of rise occurs in (0.0108) under 250 mg/L.But three phenol concentration under high sludge concentration, and the rate value of sludge medium/medium these situations of phenol concentration is all closer to, average out to 0.0094 ± 0.001/min.It is believed that this is PHA% rate of rise the highest under this experiment condition.
Improve initial sludge concentration and all have positive effect for accelerating to grow with PHA synthesis under three phenol concentration, but degree is different.Under 250 mg/L phenol concentration, the growth rate under low high sludge concentration differs about 4 times, and 1000
Then differ nearly 6 times under mg/L, illustrate that the sludge concentration in raising system helps somewhat to alleviate the toxicity of phenol.For the raising of PHA total amount, the two ratio is respectively 5 times and 13 times, illustrate that the inhibitory action that phenol synthesizes for PHA is higher than the suppression for growth, and improve sludge concentration and not only contribute to overcome the toxicity of phenol, also improve the accumulation rate of PHA total amount.For PHA total output, the combination of high phenol concentration+high sludge concentration is likely to the scheme more optimized.For improve PHA in the ratio of intracellular for, the toxicity of phenol less substantially, and the impact improving sludge concentration is the faintest.For this parameter, optimal combination is higher sludge concentration and the combination of suitable phenol concentration.Under sludge medium concentration, phenol level is more suitable at 500 mg/L, and under high sludge concentration, phenol concentration is less than 1000 mg/L.
f. Sequencing batch type operation and the highest phenol concentration:Owing to high concentration phenol is for the inhibitory action of microorganism, therefore when source waste water phenol concentration is too high when, the way of dilution can be used to reduce its toxicity.Thus invention further contemplates the effect that the phenol of Isodose adds one or more times.Fig. 5 illustrate display 1000 mg/L*1 time, 500
Mg/L*2 time and 250 situation under the conditions of mg/L*4 time, adds once (showing with arrow, abscissa-response time/min) after each phenol degrading is complete at once.
Under 250 mg/L*4 and 500 mg/L*2 both of these cases, around, and last total PHA yield is the most basically identical for the total degradation of phenol, but under conditions of 250 mg/L*4, final sludge concentration is higher and intracellular PHA proportion is relatively low.In contrast, single adds 1000 mg/L phenol and extends overall degradation time, reduces last sludge yield, and more phenol is likely to be used for PHA accumulation.The condition of 1000 mg/L*1 provides the highest total PHA yield and the highest final intracellular PHA ratio.Therefore these several different phenol addition methods cut both ways on time loss, PHA yield and productivity, overall process efficiency.If needing to shorten overall processing time, then use the mode repeatedly adding Low Concentration Phenol the most efficient；And if target is the cell obtaining and having the highest PHA ratio, then can use the disposable mode adding high concentration phenol.This kind of mode the most more meets the process requirement of high-concentration industrial-water simultaneously, simplifies integrated artistic.
Test the highest phenol concentration (2000 mg/L) further mud to be grown and the impact of PHA accumulation, now use higher initial sludge concentration.Finding that the highest phenol concentration is more strong to the inhibitory action of mud, needing nearly 7 hours could be degradable, and within this stage, sludge concentration rises to 3.5 g/L from 2.35 g/L, and PHA total concentration is from 0.43
G/L rises to 2 g/L, and intracellular PHA proportion rises to the most another attached data of 58%(from 18%).This numerical value is it is believed that close to the peak of intracellular PHA proportion under the conditions of the present invention, in conjunction with other data, it is believed that this value is between 55%-60%.
g. The impact of intracellular initial p HA% level: finding when making a general survey of each test data, when adding phenol, the initial PHA% level of intracellular has an impact possibly for integrated artistic efficiency.Therefore by many batch datas Integrated comparative, when illustrating initial sludge concentration approximation (0.9-1.2 g/L), the impact that initial intracellular PHA % level accumulates further for growth and PHA.Data are shown in Fig. 6 (abscissa-response time/min).
For generally, relatively low initial intracellular PHA level is more beneficial for phenol degrading and PHA accumulation.Under same initial sludge concentration about 1.1 g/L, in the case of initial p HA level 3.4%, the phenol of 250 mg/L can be completely degraded in 20 min, initial p HA level > 30% time degradable, need nearly 100 min.The when of it addition, initial p HA% is higher, after adding phenol, fluorescence intensity declines the most, and PHA total amount and PHA% hereafter increases the slowest.Utilize straight line portion to calculate false zero-order constant, be shown in following table:
False zero-order constant under the different initial p HA% level of table 1 compares
Therefore, in order to improve overall process efficiency, enable the phenol added to be absorbed as soon as possible, convert, to degrade, it should use initial mud relatively low for intracellular PHA % as instrument.In the technique of sequencing batch type operation, the PHA level can accumulated in mud reaches to be drawn off the most afterwards a part, as the raw material extracting PHA.And remaining part should make the hunger period that its experience is certain, the PHA consuming intracellular makes it be reduced to certain reduced levels, is beneficial to next group phenol degrading/cell growth/intracellular PHA accumulation.
h.
The impact of temperature:Temperature all has a major impact for the activity of enzymes various in cell, also has influence on the degraded of phenol, cell growth and the process of PHA synthesis.Studies have found that, under low temperature, carbon source is converted into the efficiency of PHA and has declined.Additionally having research to think, low temperature can aggravate the toxicity of phenol significantly, seriously suppresses cytoactive.The present invention uses two temperature ranges (low high), compares cell growth and the speed of PHA accumulation under the same terms.The two temperature range is respectively 7-13 DEG C and 15-23 DEG C, and some experimental conditions and result see table:
Under table 2 different temperatures, false zero-order constant compares
Comparing the mud growth under each initial phenol and sludge concentration, in two temperature ranges and PHA accumulation speed finds, under hot conditions, these three speed is both significantly higher than cryogenic conditions.This explanation is under the conditions of the present invention, and low temperature all has negative effect for cell and PHA accumulation.The two process all can be carried out at 10 ° of below C, but efficiency is greatly improved in the range of 15-23 ° of C.Therefore, in large-scale industrialization runs, certain Insulation can be added, be more beneficial for the production of stability and high efficiency PHA.
i . The impact of salt impact:
Owing to the waste water containing phenol is generally industrial source, wherein comparison of ingredients is complicated.In actual commercial production, it is possible that the component of waste water changes suddenly, such as the situation of salinity rising etc suddenly.Therefore the present invention tests salinity in normal PHA production process and raises suddenly the impact for process.Using sodium chloride as model substance, simulate the situation of salt impact in various degree, result (abscissa-response time/min) as shown in Figure 7.
From fig.7, it can be seen that the mud growth of 0% salinity (i.e. control group) and PHA accumulation have all reached higher efficiency, the phenol added is degraded complete at about 120min.To this stage, the trend of three samples essentially coincides.But after 40 minutes add NaCl, the growth of two salt impact sample mud and the accumulation of PHA all receive serious impact.Showing as OD600 the most acutely to decline, wherein concentration is that the decline of 4% salt experimental group becomes apparent from.This illustrates that the impact of high salinity has potentially affected the integrity of cell, even makes it rupture.Following period of time after 40min, the OD600 of two salt impact samples is demonstrated by the trend of stable-rising, and wherein the experimental group of 2% salinity is the rapidest.But this does not necessarily means that mud has recovered activity completely.
High salt impact also has significantly negative effect to PHA accumulation, but and not as mud grows the most substantially.Being embodied in, 2% salinity sample remains to continue accumulation PHA after adding NaCl, is about 0-40 until speed when about 3 hours
The half of min.Meanwhile in this sample, PHA% also continues to rising and is finally reached about 30%, lower by 20% than control group.But the salinity of 4% is irreversible for the impact of mud, after adding NaCl, its PHA accumulation is almost stagnated, and PHA% has declined.The degradation rate of the mud phenol after salt adding is greatly reduced, until through 6 hours, the phenol in two high salt impact groups was the most undegraded complete.Can thus be concluded that, the degraded of the growth of mud, the accumulation of PHA and phenol is affected bigger by salinity.Under experimental condition of the present invention, 2% salinity is still in the range of mud can bear, and 4% then creates irreversible infringement.Practical Project may need the salinity of waste water is necessarily controlled so that it is less than 2%.If needing to carry out phenol degrading and PHA synthesis under high salinity, then need further to carry out sludge acclimatization.
Claims (7)
1. one kind uses the method that toxic industrial pollutant phenol prepares polyhydroxyalkanoate, it is characterised in that concretely comprise the following steps:
(1) residual active sludge of use municipal sewage plant is as inoculum, to this activated sludge through certain domestication process so that it is adapt to the high phenol concentration of more than 500mg/L completely, can be by the most degradable for the phenol of this concentration complete；
(2) by the mixed bacteria mud adapting to high phenol concentration completely obtained above, cultivating in being placed on reactor, every two to three days adds phenol, nitrogen, phosphorus, calcium, magnesium, a melt quality material, maintains and cultivates；
(3), when carrying out PHA production, a mixed bacteria mud taking-up part is carried out high speed centrifugation separation every time, precipitation, obtain thickened sludge；
(4) this thickened sludge is mixed with nitrogen, phosphorus, calcium, magnesium, melt quality liquid, and add a certain amount of alkaline buffer solution, to regulate the pH value of reaction system, form the reaction mixture of a batch；
Wherein, the concentration of nutrient substance each in reaction mixture is adjusted respectively, forms certain Concentraton gradient；Adjust the initial original thickened sludge taken out, make this reaction mixture have certain initial sludge concentration；This reaction mixture is carried out aeration aerating and stirring mixing, by the intensity of regulation aeration with the dissolved oxygen concentration in change system；
(5) it is subsequently adding a certain amount of phenol so that it is there is certain initial concentration；After phenol adds, mud i.e. starts cell growth and polyhydroxyalkanoate (PHA) accumulation；Until this phenol added is degradable complete, process as a phenol and the batch of PHA accumulation.
The method preparing polyhydroxyalkanoate the most according to claim 1, it is characterized in that, after the phenol degrading of a upper batch terminates, again add a certain amount of phenol and nitrogen, phosphorus, phosphorus, calcium, magnesium, melt quality element, start next batch, to continue to improve the total amount of PHA and the ratio in intracellular.
The method preparing polyhydroxyalkanoate the most according to claim 1 and 2, it is characterised in that nitrogen element is with NH4Cl form, P elements is with K2HPO4Form, calcium constituent is with CaCl2Form, magnesium elements is with MgSO4Form, ferrum element is with FeSO4Form adds.
The method preparing polyhydroxyalkanoate the most according to claim 3, it is characterised in that in reaction system, controlling carbon-nitrogen ratio weight is 5-40(w/w)；Its computational methods are: calculated initial carbon concentration, concentration of carbon=76.6% phenol concentration by the densitometer of phenol, by this concentration compared with the nitrogen concentration of element of addition, then obtain initial carbon weight nitroxide ratio: C/N(w/w)；Controlling initial sludge concentration is 0.5-2.5 g/L；Controlling initial phenol concentration is 250-2000 mg/L；Initial ratio between phenol concentration and sludge concentration: F/M is 0.1-1.6.
5. according to the method preparing polyhydroxyalkanoate described in claim 1,2 or 4, it is characterized in that, in the reaction mixture of step (4), control phosphorus, calcium, magnesium, the concentration of ferrum are respectively 0.3-4.3 mg/L, 0-7.2 mg/L, 0-4.8 mg/L, 0-9 mg/L, and dissolved oxygen concentration is 1 mg/L-saturated concentration；Control ph is 6.5-8.5.
The method preparing polyhydroxyalkanoate the most according to claim 5, it is characterised in that the salinity controlling system is calculated as 0%-4% with NaCl.
The method preparing polyhydroxyalkanoate the most according to claim 5, it is characterised in that the temperature controlling system is 7-23 DEG C.
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| CN114250255A (en) * | 2021-10-22 | 2022-03-29 | 复旦大学 | Preparation method of high-value polyhydroxyalkanoate |
| CN114438141A (en) * | 2022-02-28 | 2022-05-06 | 复旦大学 | Preparation method for improving product value of intracellular polymer polyhydroxy fatty acid |
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