Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Assessment of cultivation parameters influencing the growth, pigment production and anti-MRSA activity of Pseudoalteromonas rubra BF1A IBRL isolated from Malaysian marine environment

Aims@#Pigments are coloured substances that exhibit important characteristics to many industries including food, textile, cosmetics, food, pharmaceutical and also aquaculture industry. Naturally derived pigments from marine bacteria do not only exhibit the tinctorial property but are also known to possess broad range of antimicrobial activities. From the industrial point of view, the necessity to obtain suitable culture conditions for maximum yield of cell growth and pigment production is of utmost importance. @*Methodology and results@#The effect of cultural conditions, including light, pH, temperature, agitation speed and size of inoculum on bioactivity of an epiphytic marine bacteria, Pseudoalteromonas rubra BF1A IBRL was studied using shake flask technology. The antimicrobial activity was determined using the Lorian method. As a result, prodigiosin pigment extract obtained from P. rubra BF1A IBRL showed inhibitory activity against the MRSA strain. Pseudoalteromonas rubra BF1A IBRL produced the highest level of prodigiosin and anti-MRSA activity (P<0.05) in Marine broth at initial pH of 7.6 incubated at dark condition at temperature of 26 °C, agitation speed of 120 rpm and 2% (v/v) (1 × 106 CFU/mL) of inoculums size. @*Conclusion, significance and impact of study@#A high correlation between pigmentation and antibacterial activity were observed anticipating that the pigment has its own antibacterial properties. The above findings supported the fact that epiphytic marine bacteria were fruitful source for pigmented bioactive compounds, and the physical parameters had significantly influence of the pigment production.

Assessment of cultivation parameters influencing the growth, pigment production and anti-MRSA activity of Pseudoalteromonas rubra BF1A IBRL isolated from Malaysian marine environment

Aims@#Pigments are coloured substances that exhibit important characteristics to many industries including food, textile, cosmetics, food, pharmaceutical and also aquaculture industry. Naturally derived pigments from marine bacteria do not only exhibit the tinctorial property but are also known to possess broad range of antimicrobial activities. From the industrial point of view, the necessity to obtain suitable culture conditions for maximum yield of cell growth and pigment production is of utmost importance. @*Methodology and results@#The effect of cultural conditions, including light, pH, temperature, agitation speed and size of inoculum on bioactivity of an epiphytic marine bacteria, Pseudoalteromonas rubra BF1A IBRL was studied using shake flask technology. The antimicrobial activity was determined using the Lorian method. As a result, prodigiosin pigment extract obtained from P. rubra BF1A IBRL showed inhibitory activity against the MRSA strain. Pseudoalteromonas rubra BF1A IBRL produced the highest level of prodigiosin and anti-MRSA activity (P<0.05) in Marine broth at initial pH of 7.6 incubated at dark condition at temperature of 26 °C, agitation speed of 120 rpm and 2% (v/v) (1 × 106 CFU/mL) of inoculums size. @*Conclusion, significance and impact of study@#A high correlation between pigmentation and antibacterial activity were observed anticipating that the pigment has its own antibacterial properties. The above findings supported the fact that epiphytic marine bacteria were fruitful source for pigmented bioactive compounds, and the physical parameters had significantly influence of the pigment production.

Production, characterization and antibacterial activity of prodigiosin pigment produced by Pseudoalteromonas rubra BF1A IBRL associated with a marine macroalgae Enteromorpha sp

Aims@#Marine bacteria are a great source of natural pigments, which can be used as colouring agent in food, textile, cosmetics and aquaculture industry to overcome the drawbacks poses by the synthetic pigments. The aim of the study is to identify the potential bio pigment producer, determine the antimicrobial susceptibilities, and characterize the pigment produced. @*Methodology and results@#In this study, the surface attached marine bacteria isolated from the surface of seaweed, Enteromorpha sp. has been identified as Pseudoalteromonas rubra BF1A IBRL through the molecular identification step. This species produced intracellular and extracellular red pigment with antibacterial activity. The susceptible bacteria include Bacillus subtilis, Bacillus cereus, Methicillin Resistant Staphylococcus aureus, Staphylococcus aureus and also Acinetobacter anitratus with inhibition zone ranges from 7.33 to 10.33 mm, whereas Minimum inhibitory concentration (MIC) values ranges from 0.055 to 8.88 mg/mL. The UV/vis analysis indicated that the maximal absorbance of ISO and DE pigment extract were at 531 and 534 nm, respectively. Based on the antimicrobial activity, the extracellular extract poses greater antibacterial activity, thus was selected as the potential pigment extract and were further evaluated. The Thin Layer Chromatography (TLC) profile of the DE extract showed one major band under visible light ((Rf = 0.87) and the bioautography analysis of the pigmented band showed positive activity against both Gram-positive and Gram-negative bacteria. The pigment in DE extract was identified as prodigiosin based on the spectroscopic properties, presumptive test and HPLC analysis. @*Conclusion, significance and impact of study@#This study highlights the dual benefits of the P. rubra BF1A IBRL pigment extract, which exhibited both tinctorial and pharmacology benefits, thus it can be act as colouring agent with own preservative value in food, textile, or cosmetics industries.

Fermentation optimization and enzyme characterization of a new ι-Carrageenase from Pseudoalteromonas carrageenovora ASY5

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.

Anti-biofilm activity of the marine bacterium Pseudoalteromonas ruthenica KLPp3 against Serratia marcescens and Vibrio alginolyticus

Aims: Pseudoalteromonas ruthenica KLPp3 is the marine Gram-negative strain isolated from the surface of mud crab at Pulau Perhentian Malaysia. In this work, the anti-biofilm activity of P. ruthenica supernatant was examined on Serratia marcescen and Vibrio alginolyticus. Methodology and results: The crude extract of P. ruthenica KLPp3 was obtained using ethyl acetate. The subminimum inhibitory concentration (MIC) of the crude extract was determined using the minimum inhibitory test. The subMIC crude extract was tested against two of the S. marcescen virulence factors, which are the swarming ability and production of prodigiosin. The crystal violet assay was used to test the anti-biofilm activity of the sub-MIC crude extract against S. marcescen and V. alginolyticus. The productions of prodigiosin were reduced by 72%. The swarming area was reduced by 56.06%. It inhibits 26.9% and 48.5% of biofilm production in S. marcescens and V. alginolyticus respectively. The crude extract was heat stable. Conclusion, significance and impact of study: Besides combating the S. marcescens virulence factor, P. ruthenica KLPp3 crude extract in sub-MIC reduces the formation of biofilm of S. marcescens and V. alginolyticus, which may find applications in biofilm inhibition and prevention.

Oxygen limitation favors the production of protein with antimicrobial activity in Pseudoalteromonas sp

This study examined the effect of dissolved oxygen concentration on the production of biomass and metabolites with antimicrobial activity of Pseudoalteromonas sp cultured at 0, 150, 250, or 450 revolutions per minute (rev. min-1). Dissolved oxygen (D.O) was monitored during the fermentation process, biomass was quantified by dry weight, and antimicrobial activity was assessed using the disk diffusion method. The bacterium Pseudoalteromonas reached similar concentration of biomass under all experimental agitation conditions, whereas antimicrobial activity was detected at 0 and 150 rev. min-1 registering 0% and 12% of D.O respectively corresponding to microaerophilic conditions. Antibiotic activity was severely diminished when D.O was above 20% of saturation; this corresponded to 250 or 450 rev. min-1. SDS-PAGE electrophoresis revealed a protein with a molecular weight of approximately 80 kilodaltons (kDa) with antimicrobial activity. Pseudoalteromonas is capable of growing under oxic and microaerophilic conditions but the metabolites with antimicrobial activity are induced under microaerophilic conditions. The current opinion is that Pseudoalteromonas are aerobic organisms; we provide additional information on the amount of dissolved oxygen during the fermentation process and its effect on antimicrobial activity.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5¨¬C, and showed 0.867 unit of o-nitrophenyl ¥â-D-galactopyranoside(ONPG) hydrolyzing activity at 4¨¬C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20¨¬C, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48 percent), C12:0 3OH (9.21 percent), C16:0 (41.83 percent), C17:0 ¥ø8 (7.24 percent) and C18:1 ¥ø7 (7.04 percent). All suthese results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20¨¬C and 7.8, respectively. The enzyme was stable at 4¨¬C for 7 days, but its activity decreased to about 50 percent of initial activity at 37¨¬C in 7 days.

Molecular cloning, characterization and enzymatic properties of a novel βeta-agarase from a marine isolate Psudoalteromonas sp. AG52

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A β-agarase gene which has 96.8 percent nucleotide identity to Aeromonas β-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant β-agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 ºC and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type β-agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas β-agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.

Extracellular proteases from the Antarctic marine Pseudoalteromonas sp. P96-47 strain / Proteasas extracelulares de la cepa marina antártica Pseudoalteromonas sp. P96-47

The extracellular protease-production capacity of 33 bacterial isolates taken from marine biotopes in King George Island, Antarctica, was evaluated in liquid cultures. The P96-47 isolate was selected due to its high production capacity and was identified as Pseudoalteromonas sp. The optimal growth temperature was 20 °C and the optimal for protease production was 15 °C. Proteases were purified from culture supernatants, developing a multiple-band profile in zymograms. They were classified as neutral metalloproteases and worked optimally at 45 °C with an Eact of 47 kJ/ mol. Their stability was higher at neutral pH, retaining more than 80% of activity at pH 6-10 after 3 h incubation at 4 °C. After 90 min incubation at 40 and 50 °C, the percentages of residual activities were 78% and 44%. These results contribute to the basic knowledge of Antarctic marine proteases and also help evaluate the probable industrial applications of P96-47 proteases.

La capacidad productora de proteasas extracelulares de 33 aislamientos bacterianos tomados de biotopos marinos en la Isla Rey Jorge, Antártida, fue evaluada en cultivo líquido. El aislamiento P96-47 fue seleccionado debido a su alta capacidad productora y fue identificado como Pseudoalteromonas sp. La temperatura óptima de crecimiento fue de 20 °C y la de producción de 15 °C. Las proteasas fueron purificadas a partir del sobrenadante de cultivo, y en los zimogramas desarrollaron un perfil de múltiples bandas. Estas proteasas fueron clasificadas como metaloproteasas neutras y se observó que trabajan óptimamente a 45 °C, con una Eact de 47 kJ/ mol. Su estabilidad fue superior a pH neutro y retuvieron más del 80% de su actividad a pH 6-10 después de 3 h de incubación a 4 °C. Luego de 90 min de incubación a 40 y 50 °C, las actividades residuales fueron 78% y 44%, respectivamente. Los resultados que se presentan en este trabajo contribuyen al conocimiento básico de las proteasas marinas antárticas y también a evaluar las probables aplicaciones industriales de las proteasas de P96-47.

Advances on Pseudoalteromonas Species and Their Extracellular Bioactive Compounds / 微生物学通报

The newly established genus Pseudoalteromonas has attracted significant interest of more and more researchers by their unique biological characteristics.In this review ,the classification and ecological distribution of Pseudoalteromonas were briefly discussed,then special emphasis placed on the extracellular bioactive agents and their bioactivity of Pseudoalteromonas.Some ideas for further studies on Pseudoalteromonas were also proposed .