Influences of Nitrogen-phosphorus Ratio on the Growth and Competition of Chlorella vulga and Anabaena sp.strain PCC

Jing WANG,Liping QIU,Shunlong MENG,,Liming FAN,Chao SONG，Jiazhang CHEN,*

1.Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,China;

2.Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences,Wuxi 214081,China

Responsible editor:Nana FAN Responsible proofreader:Xiaoyan WU

The population changes and community structure of phytoplankton,the primary producer in the marine ecological system,directly affect the structure and function of marine ecological system.There is competition in the phytoplankton,and the surroundings exert significant impacts on the competition results.The primary producer of waters is affected by the nutritional elements of nitrogen and phosphorus in the waters.Those nutritional elements are the material basis of photosynthesis and synthetic organic chemicals,among which nitrogen and phosphorus are normally considered as the factors restricting the water bloom breaking[1-3].The water bloom is often related to the supply of nitrogen and phosphorus.The nitrogen and phosphorus directly affect the assimilation rate and utilization efficiency of nitrogen and phosphorus,while the nitrogen and phosphorus concentration affect the growth of algae cell and intercellular material accumulation.Chlorella vulgaris is one of the main algae in the oxidation pond,which can provide the aerobic bacteria with oxygen,and decompose the complicated organic mass into simple inorganic matter,in order to realize the processing of wastewater[4].Meanwhile,Chlorella vulgaris,as the refined of fishes,can remove the nitrogen and phosphorus in the waters[5-6].The an-Abaena sp.is one of the main harmful algae in the nutritional waters and aquaculture waters.At present,there are many studies about Chlorella vulgaris and an Abaena sp.,but most studies are about the influences of surroundings on the single algae growth[7-9].In this paper,we discuss the competition between the Chlorella vulgaris and an Abaena sp.,in order to provide references for controlling water eutrophication.

Materials and Methods

Algae

The Chlorella vulgaris and an-Abaena sp.were purchased from the Institute of Hydrobiology,Chinese Academy of Sciences.The algae cultivation applied BG11 culture medium.After being washed with clean water,the glass apparatus was immersed in the diluted hydrochloric acid,and then rinsed with aseptic water before being baked for use.The culture condition during the experimental period was consistent with the culture condition.The sunshine intensity was 2 500 lx,and the light-darkness period was 12 h:12 h.The temperature was 25 ℃.The conical flask was shaken once every two hours during the daytime everyday,and was put still during the dark period.

Methods

In this paper,we set four concentration gradients,and there were three control groups in each concentration gradient,namely Chlorella vulgaris (C group),an Abaena sp.(A group),

Chlorella vulgaris and an Abaena sp.(CA group).Each group repeated for three times.The Chlorella vulgaris and an Abaena sp.were centrifuged for five minutes at 5 000 r/min,then we removed the supernatant,and used 15 mg/L NaHCO3for centrifugal for twice.We used non-ammonia water to dilute the water into a concentration that this experiment required.The initial inoculation density of Chlorella vulgaris and anAbaena sp.was 5× 105per/ml.We prepared 200 ml of culture medium in the 250 ml conical flask,and then put them into the constant temperature incubator where the temperature was 25 ℃,the sunshine intensity was 2 200 lx,and the light and dark ratio was 12 h:12 h(no exchange of culture medium).During the culture process,we collected water from each experiment group with a 0.45 μm apertune to measure the nitrogen and phosphorus concentration,and used NaNO3,and KH2PO4,as well as non-ammonia water to complete the nitrogen,phosphorus and water to initial level(Table1).

The number of cells

We calculated the number of algae once every 24 hours after the experiment began.The experiment ended when the number of all the algae declined,and then the number of algae on the day before the number declined would be the maximum standing crop of the specific algae.

Data analysis

The growth ratio of algaeThe formula to measure the growth ratio of algae is shown as below[10]:

Nn-1where μnis the growth ratio on the nth day; Nnis the cell density on the nth day(No./L);Nn-1is the cell density on the n-1thday(No./L);tn is the culture time of Nn.tn-1is the culture time corresponding to Nn-1.The average growth ratio (μ)was the average value of growth ratio from the beginning of the experiment to the time when the maximum existing ratio reached the highest value[11].

Fitting of growth curveThe formula(2)was the fitting of the algae growth.

where N was the algae biomass,K was the maximum biomass,r was growth rate,t was the growing time and a was the constant.

Competitive inhibition parametersThe difference form of Lotka-Volterra competition model was applied to calculation the competitive inhibition parameters[12].

where NCand NAeach represent the number of Chlorella vulgaris and an Abaena sp.NC,n-1and NA,n-1indicate the number of Chlorella vulgaris and an Abaena sp.at the tn-1moment.rCand rAeach represent the intrinsic rate of increase of Chlorella vulgaris and an Abaena sp.KC,KAshow the maximum environment capacity of Chlorella vulgaris and an Abaena sp.α and β each show the competition inhibition parameters of Chlorella vulgaris to an Abaena sp.,and the competition inhibition parameters of an Abaena sp.to Chlorella vulgaris.

The above-mentioned formula was applied to calculate all the competition inhibition parameters from the time when the mixed culture of algae reached the inflection point to the time when it met the maximum environment capacity,and the average value was considered as the estimated value of such algae[13].Considering the Logistic equation,after the inflection point of algae growth curve,the value was gradually stabilizing at the same level.The inflection point was also called the setting point of density inhibition,which means Logistic equation secondary value was the time tp value at 24:00 when N was the same was K/2,and tp equaled to a/r,where tp was the integer value.

Statistics analysis

This paper used single variation analysis method to process data,and used t test method to carry out regression significance tests.

Results and Analyses

Growth of chlorella vulgaris and an Abaena sp.at different ratios of nitrogen to phosphorus

As is shown in Fig.1,in the pure cultivation system,the influences of nitrogen to phosphorus ratio on the Chlorella vulgaris were insignificant (P＞0.05).In the mixed culture system,the influences of different ratios of nitrogen to phosphorus on the maximum biomass of two algae were significant(P＜0.05).In the pure culture system,the largest biomass of Chlorella vulgaris ranged from the largest one to the smallest one as moderate-low ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus ＞low moderatehigh ratio of nitrogen to phosphorus.In the pure cultivation system,the largest biomass of an Abaena sp.was ranged from moderate-high ratio of nitrogen tophosphorus ＞high ratio of nitrogen to phosphorus ＞moderate-low ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.There were significant differences between the moderate-high ratio of nitrogen to phosphorus and the moderate-low ratio of nitrogen to phosphorus.At the same culture time,the biomass of Chlorella vulgaris from the first to the thirteenth day was listed as low ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞moderate low ratio of nitrogen to phosphorus ＞ moderate high ratio of nitrogen to phosphorus.The biomass of an Abaena sp.ranged from moderate-high ratio of nitrogen to phosphorus,high ratio of nitrogen to phosphorus ＞moderate-low ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.In the mixed culture system,the maximum biomass of Chlorella vulgaris ranged from moderate-low ratio of nitrogen to phosphorus ＞moderate-high ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.In the mixed culture system,the largest biomass of was moderate-low ratio of nitrogen to phosphorus ＞moderate high ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.In the mixed culture system,the sequence of largest biomass of an Abaena sp.was moderate-high ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞moderate low ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.At the same culture time,the sequence of biomass of anAbaena sp.was that moderate high ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞moderate low ratio of nitrogen to phosphorus ＞low ra

tio of nitrogen to phosphorus.Table2 shows the influences of the nitrogen to phosphorus ratio on the average growth of two algae.In the pure cultivation and mixed cultivation systems,the average growth ratio of anAbaena sp.was as moderate-high ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞moderate-low ratio of nitrogen to phosphorus ＞low ratio of nitrogen to phosphorus.The average growth rate of Chlorella vulgaris was listed as low ratio of nitrogen to phosphorus ＞moderate-low ratio of nitrogen to phosphorus ＞moderatehigh ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus.The average growth ratio under mixed culture condition was listed as low ratio of nitrogen to phosphorus ＞moderatelow ratio of nitrogen to phosphorus ＞high ratio of nitrogen to phosphorus ＞moderate high ratio of nitrogen to phosphorus.According to Fig.1,in the pure cultivation system,the growth ratio of Chlorella vulgaris and an Abaena sp.at different ratios of nitrogen to phosphorus was basically similar to“S” curve,which suggested that the growth curve can be applied in the Logistic model fitting.In order to calculate the inflectiontime,Logistic equation was applied to fit the growth of Chlorella vulgaris and an Abaena sp.in the mixed culture system to get the turning-point time (Table3).In the pure culture system,the inflectiontime of anAbaena sp.was earlier than the common Chlorella vulgaris.In the mixed culture system,under different ratios of nitrogen to phosphorus,the inflectionpoint of Chlorella vulgaris was earlier than that of an Abaena sp.

Table1 The nitrogen and phosphorus concentration of different experiment groups

Table2 The average growth ratio of Chlorella vulgaris and Anabaena sp.at different

Table3 The Logistic model fitting parameters and inflection time of Chlorella vulgaris and Anabaena sp.under different nitrogen and phosphorus ratios

Table4 The competition inhibition parameters of Chlorella vulgaris and Anabaena sp.

The competition inhibition parameters of Chlorella vulgaris and an Abaena sp.

As we can see from Table4,the competition inhibition parameters of low ratio of nitrogen to phosphorus and moderate-low ratio of nitrogen to phosphorus were higher than the competition inhibition parameters of Chlorella vulgaris to an Abaena sp.When the nitrogen to phosphorus was low,the competition inhibition parameter of Chlorella vulgaris to anAbaena sp.was the maximum one,and when the nitrogen to phosphorus was moderate-high,the competition inhibition parameter of an Abaena sp.to Chlorella vulgaris was the maximum one.

Conclusions and Discussions

This study suggested that the standing rate of an Abaena sp.at moderately-high nitrogen to phosphorus was the maximum one,which means that there was one optimal ratio of nitrogen to phosphorus in the an-Abaena sp.The ratio of nitrogen to phosphorus exerted little influences on the growth of Chlorella vulgaris in the pure cultivation system,but the ratio of nitrogen to phosphorus had significant effects on the growth of Chlorella vulgaris in the mixed culture system.When the ratio of nitrogen to phosphorus was moderate-low,the standing rate of Chlorella vulgaris was the maximum.The inhibition competition parameter of the ratio of nitrogen to phosphorus was significant.When the ratio of nitrogen to phosphorus was low,the competition inhibition parameter of anAbaena sp.on the Chlorella vulgaris was the largest one.When the ratio of nitrogen to phosphorus was moderate-high,the competition inhibition parameter of Chlorella vulgaris on the an Abaena sp.was the highest one.When the ratio of nitrogen to phosphorus was low and moderatehigh,the an Abaena sp.has advantages over competition.When the ratio of nitrogen to phosphorus was moderate-high and high,the an Abaena sp.and Chlorella vulgaris were conditionally stable.

Influences of the ratio of nitrogen to phosphorus on the growth of an Abaena sp.and Chlorella vulgaris

Nitrogen and phosphorus are limiting factors in the algae growth in the natural waters.We considered the influences of nutritional salt on the growth of phytoplankton in terms of the relative concentration of nutritional salt[15].The experiment suggested that the growth rate of Chlorella vulgaris was the largest one when the ratio of nitrogen to phosphorus was 16:1,which was consistent with the study result of Ma Caihong et al.[16]The growth rate of an Abaena sp.was the largest when the ratio of nitrogen to phosphorus was 64:1,which was inconsistent with studies[17]by Wang Yachao et al.In the pure culture system,the influence of Chlorella vulgaris on the ratio of nitrogen to phosphorus was insignificant.The maximum biomass of an Abaena sp.at the nitrogen to phosphorus ratio of 64:1 was significantly different from the maximum biomass of anAbaena sp.at the nitrogen to phosphorus ratio of 16:1 and 31:1,which suggested that an-Abaena sp.can grow in an optimal nitrogen to phosphorus ratio.This had also been proved by Gu Qiahua[18].Phosphorus not only is involved in the transport of carbohydrate among different algae cells,but also directly participates in ATP metabolism[19].Studies by Xue Lingzhan et al.proved that the growth speed of an Abaena sp.rose rapidly when the phosphorus concentration rose to 150 mg/L,which raised the phosphorus concentration in the water and promote the algae growth[20].

Influences of nitrogen to phosphorus ratio on the growth of Chlorella vulgaris and an Abaena sp.

Studies prove that when the nitrogen to phosphorus ratio was low,the competition inhibition parameter of an Abaena sp.on Chlorella vulgaris was the largest one,and when the nitrogen to phosphorus ratio was moderatehigh,the competition inhibition parameter of Chlorella vulgaris on an Abaena sp.was the largest one,which suggested that the algae competition inhibition parameter was greatly influenced by nitrogen and phosphorus ratio.With different nitrogen and phosphorus ratios,both an Abaena sp.and Chlorella vulgaris were in different competition advantageous positions.Liao believed that with the addition of nitrogen,algae with different nitrogen fixation had its own advantages[21].Studies by Xu Qiujing proved that when the initial inoculation concentration was 1:1,the chlamydomonas reinhaidtii felt pressure and produced more algae toxin to inhibit the growth of another algae[22],which meant that the inhibition effect of anAbaena sp.on the Chlorella vulgaris was earlier than the inhibition effect of Chlorella vulgaris on an Abaena sp.[23-24].The fact that the growth of a certain plankton plant was limited by lack of certain nutritional salt might promote the growth of another kind of plant that had no requirements of nutritional salt.Studies by Hu Kaihui et al.proved that an Abaena sp.reproduced during its growth by assimilating nutritional materials like nitrogen and phosphorus from the external part,but also reduced the nitrogen and phosphorus content in the external environment[4].Therefore,in the mixed culture system,the growth of Chlorella vulgaris changed the ratio of nitrogen to phosphorus.When the Chlorella vulgaris was inhibited first,its competition inhibition parameter to an Abaena sp.was larger than the competition inhibition parameter of an Abaena sp.on Chlorella vulgaris.

[1]ZHAI S,YANG L,HU W.Observations of atmospheric nitrogen and phosphorus deposition during the period of algal bloom formation in northern Lake Taihu,China [J].Environmental Management,2009,44(3):542-551.

[2]STEVENSON R J,BENNETT B J,DONIELLE N J,et al.Phosphorus regulates stream injury by filamentous green algae,DO,and pH with thresholds in responses [J].Hydrobiologia,2012,695(1):25-42.

[3]ZHANG J Y,NI W M,LUO Y,et al.Response of freshwater algae to water quality in Qinshan Lake within Taihu Watershed,China [J].Physics and Chemistry of the Earth,2011,36(9/10/1):360-365.

[4]HU KH,ZHU H,WANG SH,et al.The removal effect of Chlorella vulgaris growth on nitrogen and phosphorus (小球藻对水体氮磷的去除效率)[J].Journal of Fujian Agriculture and Forestry University (Natural Science Edition) (福建农林大学学报:自然科学版),2006,35(6):648-651.

[5]YU YL(余云龙),ZOU H(邹华),ZHANG Q (张强),et al.Study on wastewater treatment by Chlorella vulgants under dark condition (黑暗条件下普通小球藻处理废水的研究)[J].Journal of Food Science and Biotechnology (食品与生物技术学报),2012,31(9):938-943.

[6]LIU SP(刘淑坡),LI F(李飞).Removal of different forms nitrogen and phosphorus in artificial wastewater by immobilized Chlorella pyrenoidosa(固定化核蛋白小球藻对人工废水中不同形态氮和磷的去除)[J].Journal of Shandong University of Technology(Natural Science Edition)(山东理工大学学报:自然科学版),2012,26(4):43-47.

[7]ZHU M,ZHU G,ZHAO L,et al.Influence of algal bloom degradation on nutrient release at the sediment water interface in Lake Taihu,China [J].Environmental Science and Pollution Research,2013,20(3):1803-1811.

[8]WANG XD(王小冬),QING BQ(秦伯强),GAO G (高光).Experimental examination on cyanobacteria bloom formation under high water temperature and strong solar irradiance(高温强太阳光照条件下蓝藻水华形成的实验研究)[J].Ecological Science (生态科学),2008,27(5):357-361.

[9]LI BJ (李宝洁).Biological effects of arsenic on Chlorella vulgaris mediated by different concentrations of phosphorus(不同磷浓度下砷对小球藻Chlorella vulgaris 的生物效应)[D].Ocean University of China(青岛:中国海洋大学),2012.

[10]Chu Z S,Jin X C,Iwami N,et al.The effect of temperature on growth characteristics and competitions of Microcystis aeruginosa and Oscillatoria mougeotii in a shallow,eutrophic lake simulator system [J].Hydrobiologia,2007,581(1):217-223.

[11]MENG SL (孟顺龙),QIU LP (裘丽萍),HU GD(胡庚东),et al.Effects of nitrogen and phosphorus ratios on the growth and competition of two bluegreen algae(氮磷比对两种蓝藻生长及竞争的影响)[J].Journal of Agro-Environment Science(农业环境科学学报),2012,31(7):1438-1444.

[12]CHEN JZ(陈家长),MENG SL(孟顺龙),HU GD (胡庚东),et al.Effects of temperature on interspecific competition between two blue-green algae(温度对两种蓝藻种间竞争的影响)[J].Chinese Journal of Ecology (生态学杂志),2010,29(3):454-459.

[13]CHEN DH (陈德辉),LIU YD (刘永定),YUAN JF (袁峻峰),et al.Experiments of mixed culture and calculation of competitive parameters between Microcystis (Cyanobacteria) and Scenedesmus (Green algae) (微囊藻和栅藻共培养实验及其竞争参数的计算)[J].Acta Ecologica Sinica (生态学报),1999,19(6):908-913.

[14]MAO H(茅华),XU H(许海),LIU ZP(刘兆普),et al.Effects of initial cell density on population competition between Skeletonema costatum and Chaetoceros curvisetus(不同起始细胞数量对旋链角毛藻和中肋骨条藻种群竞争的影响)[J].Marine Environmental Science(海洋环境科学),2008,27(5):458-461.

[15]XU H(许海),ZHU GW(朱广伟),QING BQ (秦伯强),et al.Influence of nitrogen-phosphorus ratio on dominance of bloom-forming cyanobacteria (Microcystis aeruginosa)(氮磷比对水华蓝藻优势形成的影响)[J].China Environmental Science ( 中国环境科学),2011,31(10):1676-1683.

[16]MA CH(马彩虹),MA XX(麻晓霞),MA YL (马玉龙),et al.Influences of different concentrations of N,P on the growth performance of Chlorella sp.(不同氮磷浓度对小球藻生长性能的影响)[J].Journal of Anhui Agricultural Sciences (安徽农业科学),2012,40(22):11367-11369.

[17]WANG YC (王亚超),XU HS (徐恒省),WANG GX (王国祥),et al.The impact of nitrogen,phosphorus and other environmental factors on Microcystis and An Abaena growth in Taihu Lake (氮、磷等环境因子对太湖微囊藻与水华鱼腥藻生长的影响)[J].Environmental Monitoring and Forewarning(环境监控与预警),2013,5(1):7-10.

[18]GU QH(顾启华),ZHAO L(赵林),TAN X (谭欣).Studies on competition predominance of Micryocystis aeruginosa,AnAbaena spiroides and Aphanizomeno flos-aquae (铜绿微囊藻·螺旋鱼腥藻和水华束丝藻竞争优势的研究)[J].Journal of Anhui Agricultural Sciences(安徽农业科学),2007,35(7):1990-1991,2031.

[19]KHOZIN-GOLDBERG I,COHEN Z.The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmato-phyte-Monadussubterraneus[J].Phytochemistry,2006,67(7):696-701.

[20]薛凌展,陈小晨,黄种持,等.温度和磷交互作用对铜绿微囊藻和小球藻生长的影响[J].安徽农学通报,2011,17(13):23-25,41.

[21]Liao C F H.The effect of nutrient enrichment on nitrogen fixation activity in the Bay of Quinte,Lake Ontario[J].Hydrobiologia,1977,56(3):273-279.?

[22]XU QJ(许秋瑾),GAO G(高光),CHEN WM (陈伟民).Studies on microcystin from population competition aspect(从种群竞争的角度初步研究微囊藻的产毒机理)[J].Journal of Biology (生物学杂志),2004,21(1):17-19.?

[23]LV Y(吕赟),WANG YJ(王应军),LENG X (冷雪),et al.Effects of cerium on physiological characteristics and microcystins release of an Abaena flosaquae(稀土铈对水华鱼腥藻生理特性及藻毒素释放的影响)[J].Journal of Agro-Environment Science(农业环境科学学报),2012,31(9):1677-1683.

[24]HUANG DX (黄道孝),XIAO JH (肖军华),PEI YX (裴承新),et al.Advances in the research of anatoxins(鱼腥藻毒素(Anatoxins) 研究进展)[J].Chinese Journal of Marine Drugs (中国海洋药物),2004(2):47-52.

[25]ZHENG SF(郑朔方),YANG SW(杨苏文),JIN XC(金相灿).Dynamic studies on the effect of nutrients on the growth of microcystis aeruginosa(铜绿微囊藻生长的营养动力学)[J].Environmental Science(环境科学),2005,26(2):152-156.