| Hypersaline organic wastewater such as petrol, chemical and dyeing wastewater, withcomplicated organic composition and high salinity (ions such as Cl-, Ca2+, and Na+), is difficultto treat by conventional biochemical processes. The diosgenin wastewater was a representative ofhypersaline wastewaters with high chloride ions (about above 10000mg/L) and organic pollutants(COD about above 20000 mg/L). To solve the problem of inhibitory effect of high salinity ontraditional biological wastewater treatment system, the objective of this paper was to screen anduse halophilic strains to treat diosgenin wastewater. Firstly, calcium chloride tolerant strains withhigh biodegradation capability were screened and identified. Secondly, morphology, biochemicalproperties, CaCl2-tolerant mechanisms and COD removal efficiencies of strains were studied.These results would offer the theoretical basis for the bacterial strain to be used in treating highchloride wastewaters. Finally, the bacterial CaCl2-tolerant strains were applied as the inducementto Upflow Anaerobic Sludge Bed (UASB) reactors and studied the characteristics of starting-upand running process of the reactors. The experimental results provide information about thetechnical parameters of biodegrading hypersaline wastewaters by salt-tolerant strains.The main results are as follows.1. Two novel CaCl2-tolerant bacterial strains (S616 and V430) isolated from the activatedsludge of the wastewater treatment system of a diosegenin production plant. The tolerance ofCaCl2 of strain V430 and S616 were determined. The results showed that bacterial strain V430and S616 could grow in the media contained 0-12.6％CaCl2. And they grew well in the mediacontained 0-9.0％CaCl2.2. The main biochemical, physiological features and the 16S rDNA partial sequences ofstrain V430 and S616 were determined. The results showed the strain V430 and S616 wereclosely related to Staphylococcus saprophyticus and Bacillus firmus by 98.0-99.0％similarity,respectively. Bacterial strains S616 and V430 determined in this study have been deposited in theNCBI (http://www.ncbi.nlm.nih.gov) data base under accession no. DQ355388 and no.DQ355389, respectively. And 6 invention patents were declared. Referred to the relatedresearches, there were not Staphylococcus saprophyticus and Bacillus firmus biologically treatingthe wastewater with complicated organic composition and high salinity. The two CaCl2-tolerant strains were successfully achieved, which would prove the useful bacterial resources and haveobvious application value and theoretic significance in treating the hypersaline wastewater.3. Detailed studies on COD of Diosgenin wastewater removal efficiencies of the strain S616indicated that COD removal efficiency was up to about 78.9％when degradation time, pH,temperature, and inoculation were 48h, 7-8, 30-35℃and 1ml, respectively. And for strain V430,the optimum COD removal efficiency was up to about 65.8％when degradation time, pH,temperature, and inoculation were 96h, 7-8, 30-35℃and 1ml, respectively. The results wouldoffer the theoretical basis for the bacterial strain to be used in treating high chloride wastewaters.4. Strain V430 was used for more detailed studies,on the physiological and biochemicalcharacteristics under high-salt stress and the adaptation mechanisms.The main conclusions are asfollows.(1) SEM and TEM observation on the ultrastructure of strain V430 under different CaCl2concentrations provided critical information on the surface structure of cells. When strain V430cells were incubated in the presence of high CaCl2 concentration culture medium, especially for9.0％CaCl2, there was secretion enveiling the surface of cell, making the surface irregular in theform of drapes. Moreover, the TEM images of cells grown in different CaCl2 concentrationculture media proved that the cytoplasm density of strain V430 is sparse when its cells wereincubated in the presence low CaCl2 concentration (0.2％CaCl2) medium, and the cytoplasmdensity of strain V430 becomes thicker when the CaCl2 concentration of culture mediumincreased to 1.8-9.0％CaCl2. These proved that some substances of cells such as K+, amino acid,protein, and quaternary ammonium compounds (QAC) would be accumulated under high saltconditions to accommodate the high salt environment.(2) Using both conductivity and electron microscopy, changes on the cells'wall andmembrane integrity of strains were studied, with the general non-halophilic strains of S. aureusand E.coli as the controls. The results showed that the cell membrane was the first part to sufferfrom high salt (9.0％CaCl2) stress. For non-halophile (E.coli and S. aureus), the increase in CaCl2concentration resulted in remarkably increases in membrane penetration and the integrity of cellwall was affected. But the membrane penetration of V430 was not obviously increased and itscells wall and membrane contact very well. These showed that the strain V430 has thecapabilities of keeping normal osmotic stress and preventing membrane albumen fromdestruction under high CaCl2 stress.(3) The total protein content of strain V430 cells incubated in the presence of 0.2％, 1.8％5.4％and 9.0％CaCl2 culture media showed that the total protein content increased with theCaCl2 increase in the culture medium. This showed that strain V430 had the abilities ofproducing some protein under high-salt conditions. These proteins could not only participateunder the conditions of high salinity water absorption, metabolism such as amino acids,carbohydrate and material transport, but also keep the normal physiological function of themembrane. Moreover, the total protein content was changed with increase in salt stress time and it rapidly increased to 22.38 mg·g dry cell-1 in 30min and then kept a dynamic equilibrium. Thisshowed that strain V430 could produce some proteins in a short time under high salt stress. Andsome proteins would be hydrolyzed to form amino acids which would participate in somephysiological activities. Protein synthesis and degradation could be in a dynamic equilibriumunder high salt stress.(4) Strain V430 cells could accumulate a mass of K+, free amino acids, and QAC in a shorttime when they were suffered from high salt stress. And the primary physiological andbiochemical reaction in cells was accumulation of a certain amount of potassium. And then freeamino acids (mainly glutamic acid) and QAC would be accumulated in strain V430 cells. Anappropriate amount of K+ was favorable for the synthesis of free amino acids and QAC.(5) Under high salinity stress, free amino acids (mainly glutamic acid) and QAC were themain organic osmoregulation substances of strain V430.1) The levels of free amino acids increased with increase in CaCl2 concentration of themedium. The increase in total free amino acids was mostly due to accumulation ofglutamic acid.As the salt concentration was increased from 0.2％to 9.0％CaCl2, Aspartic acid, proline,isoleucine, lysine, valine and leucine increased 1.6, 26.6, 283, 2.7, 6.7 and 44.7-fold. It wasreported that Gram-positive bacteria has a higher content of free amino acids mainly made ofglutamic acid under control (low-salt) conditions. In this study, the total free amino acids contentwhich was mainly made of glutamic acid was relatively high, up to 51.1 mgog dry cell-1 underlow salt stress (0.2％CaCl2), showing the compositional characteristics Gram-positive bacteriatypically have. The accumulation of proline or other free amino acids was observed in somemicroorganisms exposed to high salinity. Gram-positive bacteria were reported to accumulate theneutral amino acids proline in response to increasing salt stress. Increase of glutamic acid wasosmotically induced in numerous gram-negative bacteria. However, in this study, aspartic acid,proline, isoleucine, lysine, valine and leucine were accumulated in strain V430 of Staphylococcussaprophyticus, as the main osmoprotectant to make the strain acclimated to high CaCl2conditions, which is different from the behavior of ordinary Gram-positive bacteria. Theoccurrence of isoleucine, lysine, valine and leueine as inducible compatible solutes inStaphylococcus saprophyticus has not been reported in previous studies. Therefore, in terms ofresponse to salt stress, strain V430 of Staphylococcus saprophyticus has several specific featuresthat have not been found in other gram-positive bacteria. Moreover, strain V430 cells couldaccumulate free amino acid in a short time when they were suffered from 9.0％CaCl2 salt stress.The total intracellular free content could be at 120 min to 168.4 mgo g dry cells-1, up to 73％ofthe content of stable-phase cells incubated in 9.0％CaCl2 medium.2) The QAC content of strain V430 cells incubated in different CaCl2 concentration culturemedium showed that the OD365nm value of strain V430 was up to 0.172 in the presence of 0.2％CaCl2 culture medium, but it would rise to 0.86, 3.20, and 6.03 times when the CaCl2concentration in the mediums was 1.8％,5.4 and 9.0％, respectively. The remarkable increase in the concentration of QAC with the CaCl2 concentration increased showed that QAC played animportant role in the osmorgulation process of CaCl2-tolerant strain V430. The QAC content ofstrain V430 cells under 9.0％CaCl2 stress for 30min,60min,90min,120min and 150min showedthat the QAC content of strain V430 would increased with the salt-stress time increase. When thesalt-stress time was only up to 150min, the levels of intracellular QAC could be 5.9-folds of thecompared sample. These showed that strain V430 cells could accumulate QAC in a short timewhen they were suffered from high salt stress.(6) The total soluble sugar content of strain V430 cells incubated in the presence of 0.2％,1.8％5.4％and 9.0％CaCl2 culture medium showed that the levels of intracellular soluble sugarincreased with increase in CaCl2 concentration of the medium. But the total soluble sugar contentof strain V430 cells incubated in the presence of 9.0％CaCl2 culture medium would increase first,and then decrease. These carbohydrates could be decomposed as a carbon and energy sources ofDNA and protein synthesis.(7) The extracellular secretions and Ca2+ of strain V430 cells suffering from 9.0％CaCl2stress could not change rapidly in a short time like K+, glutamic acid, and QAC. Therefore, Ca2+accumulation and extracellular secretion of strain V430 cells grown in the presence of 9.0％CaCl2 medium result from the strain V430's acclimatization to the high salt environment afer along time.(8) Under high salinity stress, strain V430 could accumulate osmoregulation by synthesisand uptake from the medium. The increase in K+ was the result of taking up from the medium,but the increase in Glutamate and QAC was the result of internal synthesis. The accumulation ofisoleucine and lysine could only take up from the external environment (medium).5. To study the applicability of the salt-tolerant strains in biological treatment of wastewaterswith high contents os salinity and organic pollutants, two sets of UASB reactors (A and B) wereconstructed and the bacterial CaCl2-tolerant strains were used as the inducement to the Reactor B.Meanwhile, the other UASB (Reactor A) which not added the CaCl2-tolerant strains was used asa control. The characteristics of starting-up and running process of the two reactors were studied.The results are as follows.(1) Inducement of salt-tolerant strains had not only improved the COD removal efficiency,but also shortened the start-up time. When start-up time was 23 days, the COD removalefficiency of Reactor A was up to 88.57％, but for Reactor B, COD removal efficiency was ashigh as 95.29％in 14 days.(2) The manner of gradually increasing Volume Loading Rate (VLR) and salinity couldcontribute to the taming of sludge. When the average VLR and chloride ions was 1.99 kgCOD/(m3·d), and 9000-10000mg/L, respectively, COD removal efficiency of the reactor A andreactor B was 90.5％and 98.99％, respectively.(3) The added salt-tolerant strains could improve the sludge activities and enhance theanti-VLR capacity. When the OLR was enhanced from 2.5 kgCOD/(m3·d) to 4.5 kgCOD/(m3·d) but kept constant the salinity ([Cl]=10000-12000mg/L), COD removal efficiency of the ReactorA had obviously changed from 77.51％to 89.72％with OLR enhanced. However, the CODremoval of Reactor B remained relatively stable with an average of 93.94％removal of COD.Meanwhile, the chloride ions average removal of A anaerobic system was up to 10.07％, and15.44％for B anaerobic system.(4) When the chloride ions of influent water was between 9000 mg/L and 14000mg/L, slightinhibition was observed on microorganisms of Reactor A. But after the sludge was beingaccommodated, COD removal efficiency of Reactor A could still keep 87％. And under theseconditions, Reactor B had a visible COD removal efficiency with the average 96.32％.(5) When the chloride ions of influent water was between 17000 mg/L and 22000mg/L,which has produced a moderate inhibition of A sludge system. And the COD removal efficiencyof Reactor A dropped to below 70％. At the same time, Reactor B had not been significantlyinhibited the activity of sludge. Appropriate salinity had been contributed to the microorganismsbreeding of Reactor B. The average COD removal rate was 92.95％.(6) When the chloride ions of influent water was 28000mg/L, which has produced seriousinhibition on microorganisms of Reactor A. COD removal efficiency has been reduced to lessthan 30％and it had only restored to 55％after 24 days recovery. But for Reactor B, the sludgecould still maintain good activities and metabolic degradation capability, the average CODremoval was still kept about 84.41％.The major innovations achieved in this thesis are as follows:1. Two novel CaCl2-tolerant bacterial strains (S616 and V430) capable of biologicallydegrading refractory organics in wastewaters were successfully obtained. The main biochemical,physiological features and the 16S rDNA partial sequences of strain V430 and S616 weredetermined. The results showed that bacterial strain V430 and S616 were closely related toStaphylococcus saprophyticus and Bacillus firmus by 98.0-99.0％similarity, respectively. Andthe performances of strain S616 and V430 biodegrading the COD of Diosgenin wastewater werestudied.2. The soluble sugar, free amino acids compositions, protein, K+, Ca2+,Mg2+ and QACcontent (OD365nm) of strain V430 under high CaCl2 concentration stress were firstly determined.Make sure the free amino acids (mainly glutamic acid, proline, isoleucine and lysine) and QACwere the main organic osmoregulation substances of strain V430.3. The ultrastructure of strain V430 under different CaCl2 concentrations was firstly observed.
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