Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Cultivation of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura: mass production of zygote-derived seedlings under commercial cultivation conditions, a case study experience

Mariculture of the brown alga Hizikia fusiformis (Harvey) Okamura as an export-oriented human food has been there more for than 20 years in China. It is now one of the five major farmed algal species along the Chinese coast. Stable and sufficient supply of young seedlings for scaling up the cultivation has been a problem throughout the farming history of this species due to the unique dioecious life cycle and relatively short time window of sexual reproduction in nature. These two factors led to a practical difficulty in obtaining zygotes at identical developmental stage in viable amounts for seedling production. A key solution to this problem is to control the synchronization of the receptacle development and to realize the simultaneous discharge of male and female gametes, such that the fertilization rate could be greatly enhanced. Focusing on one of the farmed populations in this report, we present our results on mass production of seedlings using the synchronization technique on a large scale performed in 2007. Totally 5.5 hundred million embryos were obtained from 100 kg female sporophytes. The seedlings were raised up to 3.5 mm in length in greenhouse tanks over a month and were further grown in open sea for over 3 months at two experimental sites. The success of mass production of seedlings in this alga helped to lay the basis for future trials in other species in the genus of Sargassum that have identical life cycle.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Study on the photosynthetic performances of Enteromorpha prolifera collected from the surface and bottom of the sea of Qingdao sea area

In this work, the photosynthetic performances of Enteromorpha prolifera thalli collected from the surface and bottom of the sea of Qingdao sea area were studied with chlorophyll fluorescence and oxygraph technology. The samples with the highest photosynthetic activity among their kinds, the floating thalli from the sea surface of the south of the Qingdao Olympic Sailing Center and the sedimentary thalli from the mud surface of the bottom Tuandao bay, were chosen as representatives of surface thalli and bottom thalli, respectively. The results showed that the maximal PSII quantum yield of the floating thalli was significantly lower than the normal level although their photosynthetic activities were relatively high; the photosynthetic potential of the thalli form the mud surface was extremely low. Thus, it is indicated that the floating thalli are seriously stressed by their environment and the thalli from the mud surface are already dead or are dying. On the other hand, the results of the laboratory cultivation showed that the sedimentary thalli cannot regain normal photosynthetic activity even under normal illumination conditions. Thus, the thalli from the mud surface cannot become reproductive source of the alga even if they can reach sea surface again. Therefore, a preliminary conclusion can be reached that, up to mid-July 2008, the environmental conditions of the Qingdao sea area are not suitable for the growth of the alga E. prolifera and for this reason the biomass of E. prolifera, in the area, could be declining.

Cultivation of the brown alga Sargassum horneri: sexual reproduction and seedling production in tank culture under reduced solar irradiance in ambient temperature

As a large conspicuous intertidal brown alga, individuals of Sargassum horneri can reach a length of more than 7 m with a fresh weight of 3 kg along the coasts of the Eastern China Sea. The biomass of this alga as a vital component in coastal water ecology has been well documented. In recent years, a steady disappearance of the algal biomass along the once densely populated coastal areas of the Eastern China Sea has drawn attention in China. Efforts have been made to reconstruct the subtidal algal flora or even to grow the alga by use of long-lines. As part of the efforts to establish an efficient technique for producing seedlings of S. horneri, in this investigation a series of culture experiments were carried out in indoor raceway and rectangular tanks under reduced solar irradiance at ambient temperature in 2007-2008. The investigation demonstrated that: (1) sexual reproduction of S. horneri could be accelerated in elevated temperature and light climates, at least 3 months earlier than in the wild; (2) eggs of S. horneri had the potential to be fertilized up to 48 h, much longer than that of known related species; (3) suspension and fixed culture methods were both effective in growing the seedlings to the long-line cultivation stage; and (4) the life cycle of S. horneri in culture could be shortened to 4.5 months, thus establishing this alga as an appropriate model for investigating sexual reproduction in dieocious species of this genus.

Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process

We have developed a two-stage growth one-step process for cultivation of Haematococcus using a self-designed system that mimics an open pond in the natural environment. The characteristics of this process are green vegetative cell growth and cysts transformation and pigment accumulation that proceed spontaneously and successively in one open photobioreactor. Four strains of Haematococcus (H. pluvialis 26; H. pluvialis 30; H. pluvialis 34; H. pluvialis WZ) were cultured in this imitation system for a duration of 12 days. The changes in cell density and medium pH were closely monitored, and the astaxanthin content and yield of the four Haematococcus strains were measured at the end of 12 days of cultivation. Two of the strains, H. pluvialis 26 and H. pluvialis WZ, were selected as strains suitable for mass culture, resulting in the astaxanthin yield of 51.06 and 40.25 mg L-1 which are equivalent to 2.79 and 2.50% of their dry biomass respectively. Based on the laboratory work, 6 batch cultures of H. pluvialis WZ were conducted successfully to produce astaxanthin in two 100 m(2) open race-way pond by two-stage growth one-step process. The astaxanthin content ranged from 1.61 to 2.48 g 100 g(-1) dry wt., with average astaxanthin content of 2.10 g 100 g(-1) dry wt. Compared with the one-stage production of astaxanthin based on continuous culture, the superiority of our process is that it can accumulate much more astaxanthin in red cysts. Compared with two-stage production of astaxanthin, the advantage of our process is that it does not need to divide the production process into two parts using two bioreactors. The presented work demonstrates the feasibility for producing astaxanthin from Haematococcus using a two-stage growth one-step process in open pond, culture systems that have been successfully used for Spirulina and Chlorella mass culture. The future of Haematococcus astaxanthin production has been also discussed. (C) 2009 Elsevier B.V. All rights reserved.

Optimal light regime for the cultivation of transgenic Laminaria japonica gametophytes in a bubble-column bioreactor

Fluctuating light intensity had a more significant impact on growth of gametophytes of transgenic Laminaria japonica in a 2500 ml bubble-column bioreactor than constant light intensity. A fluctuating light intensity between 10 and 110 mu E m(-2) s(-1), with a photoperiod of 14 h:10 h light:dark, was the best regime for growth giving 1430 mg biomass l(-1).

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: enhanced seedling production in tumbled culture

The reuse of holdfasts for regeneration of young seedlings or using wild juvenile plants as the seedling source has played the major role in commercial cultivation of the brown alga Hizikia fusiformis in East Asia over the past 20 years. The possibility of employing zygote-derived germlings for producing seedlings has been discussed in the literature, but has not yet become a reality. Three main obstacles have limited the use of zygotes as a main source of seedlings, (1) the dioecious nature of the algal life cycle which may lead to asynchronous male and female receptacle development and thus different timing of egg and spermatozoa expulsion, (2) the low attachment rate when using zygote-derived germlings with developed rhizoids from wild parental plants for seeding production, and (3) the problem of culturing young germlings in regions where water temperature is high in summer. In this investigation, shifting the timing of receptacle formation earlier than in nature was performed by tumbling the algae in a long-day tank (16-h light per day). Synchronization of egg and spermatozoa expulsion and thereafter fertilization were conducted in indoor tanks. Receptacle formation in constant long days could be shifted by 20 days earlier than in plants cultured on long lines in the open sea, or I month earlier than in plants growing on intertidal rocks. Synchronized expulsion of eggs and spermatozoon led to a high rate of fertilization. This was achieved by tumbling the male and female receptacle-bearing branchlets in the same tank at low density in high irradiance. In two independent trials, a total of 1,400,000 zygote-derived germlings were obtained from 620 g (fresh weight) female sporophytes. The germlings shed from the receptacles were at an identical developmental stage indicating high synchronization of expulsion of eggs and spermatozoon followed by fertilization. Approximately 63% ( +/-9.6%) of the germlings were shed from the receptacle between 16 and 24 It after fertilization and 20% ( +/-11.9%) remained on the receptacle for 3 days after fertilization. Germlings were seeded on string collectors before rhizoids started to elongate and the attachment efficiency was enhanced. Young seedlings reached 800 ( +/-50) mum in length in 25 days at 25 degreesC before they were transferred to open sea cultivation. These results provide the basis of a practical way of seedling production by use of zygote-derived germlings in the commercial cultivation of Hizikia fusiformis. (C) 2004 Elsevier B.V All rights reserved.

Tank cultivation of the red alga Palmaria palmata: Effects of intermittent light on growth rate, yield and growth kinetics

Tank cultivation of marine macroalgae involves air-agitation of the algal biomass and intermittent light conditions, i.e. periodic, short light exposure of the thalli in the range of 10 s at the water surface followed by plunging to low light or darkness at the tank bottom and recirculation back to the surface in the range of 1-2 min. Open questions relate to effects of surface irradiance on growth rate and yield in such tumble cultures and the possibility of chronic photoinhibition in full sunlight. A specially constructed shallow-depth tank combined with a dark tank allowed fast circulation times of approximately 5 s, at a density of 4.2 kg fresh weight (FW) m(-2) s(-1). Growth rate and yield of the red alga Palmaria palmata increased over a wide range of irradiances, with no signs of chronic photoinhibition, up to a growth-saturating irradiance of approximately 1600 mumol m(-2) s(-1) in yellowish light supplied by a sodium high pressure lamp at 16 h light per day. Maximum growth rate ranged at 12% FW d(-1), and maximum yield at 609 g FW m(-2) d(-1). This shows that high growth rates of individual thalli may be reached in a dense tumble culture, if high surface irradiances and short circulation times are supplied. Another aspect of intermittent light relates to possible changes of basic growth kinetics, as compared to continuous light. For this purpose on-line measurements of growth rate were performed with a daily light reduction by 50% in light-dark cycles of 1, 2 or 3 min duration during the daily light period. Growth rates at 10degreesC and 50 mumol photon m(-2) s- 1 dropped in all three intermittent light regimes during both the main light and dark periods and reached with all three periodicities approximately 50% of the control, with no apparent changes in basic growth kinetics, as compared to continuous light.

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 degreesC and light intensity of 40 mumol m(-2) s(-1) for 6 days. With aeration at 1 l min(-1), the airlift reactor yielded higher growth rate (0.12 mg DW ml(-1) d(-1)) than a bubble column (0.08 mg DW ml(-1) d(-1)). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.

Translocation of assimilates in Undaria and its cultivation in China

Undaria cultivation on a commercial scale began in China only in the last decade. Today, Undaria pinnatifida is the main species under cultivation concentrated in two provinces, Liaoning and Shandong. The annual production in the early nineties was 8000-13 000 tons dry weight, which is two or three times the pre-1980 figures. The raft cultivation method maintaining the alga at the desired depths generally ensures the light saturated rate of photosynthesis on clear days, and enhances production. Under the cultivated condition, the calculated annual primary productivity of this alga is 160 g C m(-2) y(-1). Translocation of C-14-labelled photoassimilates in rapidly growing sporophyte of Undaria pinnatifida was studied in the open sea. Samples from different parts of the blade with counterparts exposed to tracer ((NaHCO3)-C-14) showed that the translocation that occurred mainly from the tip of the blade to the growing region had obvious source-sink relationship. It took 20 minutes to translocate the labelled photoassimilates from the epidermis, via cortex, to the medulla of the midrib, where rates of translocation averaging 42-48 cm h(-1) were observed in the open sea. Production experiments of tip-cutting of the blades showed an increased production of 9%.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.