碱法草浆造纸蒸煮黑液厌氧生物降解性及其主要微生物类群研究

造纸行业是造成我国水环境有机污染物的重要污染源之一，其水污染的特点是小厂多、草浆多、工艺落后、污染扩散面广、造成废 水排放量大，每年排放的废水量约39亿立方米，占全国工业废水排放量的1/6，其中有机污染物（以BOD5计）160万吨左右，约占全 国工业废水中有机污染物总量的1/4。尤以占全国制浆造纸行业90%以上的碱法草浆造纸厂的蒸煮黑液量大面广，除含有机物外，还 含有木质素、残碱、硫化物、氯化物等污染物，属于PH值高、色度深、难于治理的高浓度有机废水，对水体污染特别严重，各地要 求治理呼声很高，急待研究并尽快找出各种有效的治理途径。对于碱法草浆蒸煮，黑液高浓度废水的治理，有各种方法，根据国内 的研究进展和我们已有试验工作表明，最经济有效，具有实用价值，在生产上可获得成功是厌氧处理法。近10多年来，国外关于高 效厌氧处理技术研究进展迅速，并出现了多种多样的工艺设备，如高效厌氧生物反应器，并在实用化方面取得了很大成绩，建立了 生产性装置，达到了高负荷运行，效果良好。本试验是根据我们已有研究基础，针对我国国情，对小型制浆造纸厂水污染防治除了 开发碱回收及各种综合利用技术外，要特别加强废水(废液)实用技术研究的指导思想，本试验采用改进型的上流式厌氧污泥床反应 器，设计了两种试验方案，通过试验结果如下。1. 试验方案I—碱法草浆黑液酸化和厌氧发酵I号UASB反应器动态模型试验结果表 明：(1). 采用中温35℃±1℃高效厌氧反应器USAB内装有填料(陶粒)和三相分离器，具有保持高浓度生物量和防止污泥流失的特点 ，污泥浓度Vs 可达30%以上，因而具有高效、节能、产能、滞留期短的优点，当进水CODcr在7500-10000mg/l，HRT由7天缩短到3天 ，有机容积负荷在1.22gCODcr/l·d-3.43gCODcr/l·d时，CODcr平均去除率可达55%-45.5%，最高CODcr去除率可达60.2-63.5%， BOD5去除率可达75.9-83.2%,沼气容积产气率可达0.29-0.67l/l·d，每克CODcr转化为沼气产率达0.39-0.48l/gCODcr·d，CH4含量 65.8-75.5%。厌氧发酵出水再用化学法进行后处理脱除难降解的木质素，CODcr总去除率达80%以上。(2). 动态试验结果表明：采 用酸化—厌氧发酵处理黑液工艺合理，技术路线可行。2. 试验方案II—黑液用化学法(Hcl)去除木质素进行厌氧发酵，II号UASB反 应器动态模型试验结果表明：(1). 采用中温35℃±1℃高效厌氧反应器UASB(内有软填料)，当进水CODcr7000-13000mg/l左右，HRT 由6天缩短到1天，有机负荷由0.98gCODcr/l·d增加到11gCODcr/l·d时，COD平均去除率均可稳定在70-77%，BOD5去除率为87.3- 93.1%，沼气容积产气率0.21-2.6l/l·d，每克CODcr转化为沼气产率为0.39-0.48l/gCODcr·d，高的可达0.53l/gCODcr·d，转化 率较高，CH4含量63-70%。(2). 试验证明碱法草浆黑液物化预处理—厌氧发酵处理的技术路线也是可行的，工艺合理、效果较好。 在有条件的工厂可采用。3.厌氧发酵阶段几大类群微生物计数表明：(1). 当发酵工艺和运行处于相对稳定状态时，微生物群体的 组成也达到相对的稳定，各类微生物之间保持动态平衡关系。当产乙酸菌的数量为107-108个/ml时，产甲烷菌的数量为105-106 个/ml，当产乙酸菌数量为106-107个/ml时，产甲烷菌的数量为103-105个/ml。(2).稳态运行条件下，黑液预处理为甲烷发酵创造 了有利的生态环境，获得了较好的处理效果和较高的COD转化为沼气的产率0.39-0.48l/g·CODcr·d，反应器中形成较为稳定而数 量较下水污泥中高1-2个数量级的厌氧发酵微生物区系组成。这一结果为黑液厌氧发酵提供了微生物理论依据。Paper industry is one of the important pollution source of water environment in our country. Its character of water pollution is many small factories, much grass pulp, disadvantageous technique, large preading area of pullution. Its effluent makes up 1/6 of whole country's industry wastwater. Its organic pollutant accounts 1/4 of whole country's. Alkaline grass paper pulp effluent with pollutants such as ligoin, remaining alkali sulfide, chloride besides organic material, is a kind of high concentrate organic wastewater which has high PH walug, dark colour and is difficult in treatment. There is urgent require to find ways to treat the wastewater. There are different ways to treat alkaline paper grass pulp effluent. According to the research advances and our experiment work, the most economical and useful way is anaerobic degradation which was advanced quick in last ten years. In the control of waste water of small pulp paper mill, the study of wastewater utilization technology should be emphasized, besides alkaline retrieving and different kinds of comprehensive utilization technology. Our experiment used modified UASB(Upflow Anaerobic Sludge Blanket Reactor). Two kinds of plan were disgned. The results are lined below. 1. The first experiment plant-aciding black pulp effluent and methanogenic digestion. The dynamic model experiment results of I-UASB reactor showed: (1)The mesophilic(35℃±1℃)high effect UASB reactor having haydite and threee state seperation in it had the character of keeping high bioimass concentration and preventing losss of sludge. It had advantages of high effect, energe saving, energe prodcing and short HRT(Hydroulic retention time). When the influent COD was 7500-10000mg, HRT was shortened from 7 days to 3days, organic loading rate was 1.22g-3.43COD/l· d, the average COD removal efficiency was 55%-45%. The highest COD efficiency was 60.2-63.5%, BOD removal of 75.9 -83.4% was achieved. Biogass production rate were up to 0.29-0.67l/l·d. Biogass converted efficiency from every gram of COD could reach 0.39-0.48l/gCOD·d. Methane content was 65.0-75.5%. Chemical method was used to deplate lignin in anaerobic digestion effluent. Total COD removal efficiency could be more than 80%. (2)Using aciding annaerobic digestion to treat the black effluent was reseanable in technique and technology. 2. The second experiment plan-anaerobic digestion was used after the chemical method was used to deplate lignin in the black effluent. The result of dynamic experiment of II-UASB reactor showed: (1)High effect mesophilic (35℃±1℃)UASB reactor having soft slaffing in was used. When influent COD was about 7000-13000mg/l, HRT was shortened from 6 days to 1 day and organic loading rate was increased from 0.90 to 11g COD /l·d, average COD removal efficiency remained stable on 70-77%. BOD, removal efficiency was between 87.3-93.1%. Biogass production rate was 0.2-2.6l/l ·d .Biogass converted efficiency from a gram of COD was 0.39-0.481/gCOD·d with the high value of 0.53l/gCOD·d. Methane content was 63-70%. (2)The way that using physical, chemical Pre-treatment-anaerobic digestion to treat alkaline black effluent is feasible and can be used in some factories when the condition exists. 3. Counting of several class of microoganisms in anaerobic digestion stage showed: (1)As the disgestion was in stable motion, the compositon of microorganic colony could get relative stable. Dynamic balance was remaining among different kinds of microorganism such as methanogenic bacteria, Acidogenic bacteria, sulfate reducing bacteria, and heterotrophic bacteria. (2)Under stable motion, the pre-treatment of black effluent produced favourable eco-enviroment for methanegenic digestion. Good treatment effect and high biogass convertent efficiency from COD(0.39-0.48l/g·COD· d)were gotten. Some stable and high quantity(10-100times more than sewage sludge)microorganism colony were formed in the reactor. This result provided theoretical basis for anaerobic digestion of black effluent.

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

Melatonin in Chinese medicinal herbs

Melatonin is a highly conserved molecule that not only exists in animals, but also is present in bacteria, unicellular organisms and in plants. Since melatonin is an antioxidant, in plants melatonin was speculated to protect them from intrinsic and environmental oxidative stress. More importantly, melatonin in edible plants inevitably enters animals and human through feed and food. In this study, more than 100 Chinese medicinal herbs were analyzed using the methods of solid phase extraction and HPLC-FD on-line with MS to determine whether melatonin is present in these commonly used herbs. Melatonin was detected in majority of these plants. Sixty-four of them contain melatonin in excess of 10 ng per gram dry mass. Melatonin levels in several herbs are in excess of 1000 ng/g. It is well known that normal average physiological plasma levels of melatonin are only 10-60 pg/mL. These high level-melatonin containing plants are traditionally used to treat diseases which presumably involve free radical damage. The current study provides new information concerning one potentially effective constituent present in a large number of medicinal herbs. The results suggest that these herbs should be reevaluated in reference to their nutritional and medicinal value. (C) 2003 Elsevier Science Inc. All rights reserved.

Effects of cerium on the microstructure and mechanical properties of Mg–20Zn–8Al alloy

Mg-20Zn-8Al-xCe(x=0-2 wt.%) alloys were prepared by metal mould casting method, the effects of Ce on the microstructure and mechanical properties of the alloys were investigated. The results showed that the dendrite as well as gram size were refined by the addition of Ce, and the best refinement was obtained in 1.39% Ce containing alloy.

Kinetic analysis of interaction between lipopolysaccharide and biomolecules

Lipopolysaccharide ( LPS) is a major component of the outer membrane of all gram-negative bacteria. It is a heat-resistant toxin which can cause toxic shock in animals. LPS interacts with some biomolecules and triggers its toxic reaction. In this study, the interaction between LPS from Salmonella Minnesota and some biomolecules using syrface okasnib resibabce ( SPR) biosensor. biomolecules were imobilized on CM5 sensor-chip suing amion coupling method and LPS was injected over the immobilized surfaces.

Monodisperse mesoporous superparamagnetic single-crystal magnetite nanoparticles for drug delivery

In this contribution, we report a facile, gram-scale, low-cost route to prepare monodisperse superparamagnetic single-crystal magnetite NPs with mesoporous structure (MSSMN) via a very simple solvothermal method. The formation mechanism of MSSMN is also discussed and we think that Ostwald ripening probably plays an important role in this synthesis process. It is also interestingly found that the size and morphology of mesoporous Fe3O4 NPs can be easily controlled by changing the amount of NaOH and 1,2-ethylenediamine (ETH). Most importantly, the MSSMN can be used as an effective drug delivery carrier. A typical anticancer drug, doxorubicin (Dox), is used for drug loading, and the release behaviors of Dox in two different pH solutions are studied. The results indicate that the MSSMN has a high drug loading capacity and favorable release property for Dox; thus, it is very promising for the application in drug delivery.

Effects of cerium on the microstructure and mechanical properties of Mg-20Zn-8Al alloy

Mg-20Zn-8Al-xCe(x=0-2 wt.%) alloys were prepared by metal mould casting method, the effects of Ce on the microstructure and mechanical properties of the alloys were investigated. The results showed that the dendrite as well as gram size were refined by the addition of Ce, and the best refinement was obtained in 1.39% Ce containing alloy. The main phases in the as cast alloys were alpha-Mg and tau-Mg-32 (Al, Zn)(49), and Al4Ce phase was found in the alloys contained more than 1.39% Ce. The addition of Ce improved the mechanical properties of the alloys. The strengthening mechanism was attributed to grain refinement and compound reinforced.

Concentration-dependent behavior of nisin interaction with supported bilayer lipid membrane

Nisin is a positively charged antibacterial peptide that binds to the negatively charged membranes of gram-positive bacteria. The initial interaction of the peptide with the model membrane of negatively charged DPPG (dipalmitoylphosphatidylglycerol) was studied by cyclic voltammetry and a.c. impedance spectroscopy. Nisin could induce pores the supported bilayer lipid membrane, thus, it led to the marker ions Fe(CN)(6)(3-/4-) crossing the lipid membrane and giving the redox reaction on the glassy carbon electrode (GCE). Experimental results suggested that the pore formation on supported bilayer lipid membrane was dependent on the concentration of nisin and it included three main concentration stages: low, middling, high concentration.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.