Bioresource Technology 97 (2006) 1843–1849
0960-8524/$ - e front matter © 2005 Elvier Ltd. All rights rerved.
doi:10.1016/j.biortech.2005.08.021
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Abstract
A unicellular alga displaying a high growth rate under heterotrophic growth conditions was isolated from soil and identi W ed as Chlo-rella sorokiniana . The optimal temperature for growth was 35°C and the optimal pH was 6.0–7.0. Gluco, sucro, galacto, malto,and soluble starch rved as carbon sources supporting growth under dark conditions. The cell yield was 50g/l (wet weight) in a hetero-trophic medium containing 3% gluco. Isolated unicellular algae were highly resistant to heavy metals such as Cd 2+, of which the mini-mal inhibitory concentration was 4mM. Algae were capable of taking up the heavy metal ions Cd 2+, Zn 2+ and Cu 2+ at 43.0, 42.0 and 46.4 g/mg dry weight, respectively. Growth inhibition of Oryza sative shoots by 5ppm Cd 2+ in hydroponic medium was completely pre-vented by the addition of 0.25mg of wet Chlorella cells. The results indicated that this isolate was potentially uful for phytoremedia-tion by preventing environmental dispersion of heavy metals.© 2005 Elvier Ltd. All rights rerved.
Keywords:Bioremediation; Cadmium; Chlorella sorokiniana ; Green algae; Heterotrophic growth; Phytoremediation
1. Introduction
Many metal ions are esntial as trace elements, but at higher concentrations, they become toxic. Heavy metals are di Y cult to remove from the environment and are ultimately indestructible, unlike many other pollutants that can be chemically or biologically degraded (Ozaki et al., 2003).Today, heavy metals constitute a global environmental haz-ard. For example, environmental pollution by Cd 2+, arising mainly from mining, smelting, dispersal of wage sludge (Hutton, 1983), and the u of phosphate fertilizers (Cris-anto Herrero and Lorenzo Martin, 1993) is increasing.Microorganisms could be ud to clean up metal contami-nation by removing metal from contaminated water and waste streams, questering metals from soil and diments,or solubilizing metals to facilitate their extraction. Bacteria and higher microorganisms have developed resistance to
toxic metals and are able to make them innocuous. Micro-organisms respond to heavy metals using various defen systems, such as exclusion (Ortiz et al., 1992), compartmen-talization (Valls et al., 2000), complex formation (Wang et al., 1997), and synthesis of binding proteins, such as metallothio
neins (Adamis et al., 2004). Microorganisms with unique abilities such as metal absorption, accumula-tion or resistance can be identi W ed among naturally occur-ring organisms. Alternatively, the systems can be utilized in engineering bacteria for remediation of polluted waters and soils. Thus, the u of microorganisms for decontami-nating heavy metals has attracted growing attention becau there are veral problems associated with pollu-tant removal using conventional methods. Bioremediation strategies have been propod as an attractive alternative owing to their low cost and high e Y ciency (Mejare and Bülow, 2001).
Heavy-metal-resistant microorganisms show us possible methods to prevent environmental contamination. The newly discovered metal questering properties of certain
*
Corresponding author. Tel./fax: +81 985 58 7218.
E-mail address: a04109u@cc.miyazaki-u.ac.jp (N. Yoshida).
1844N. Yoshida et al. / Bioresource Technology 97 (2006) 1843–1849
types of fungi (Svoboda and Kalac, 2003) and algae (Chai-suksant, 2003) hold considerable promi.
商务英语翻译 Heavy metals can be eliminated from polluted environments by utilizing their natural heavy metal disposing abilities. The objective of this study was thus to isolate and characterize a cad-mium-tolerant eukaryotic microorganism from soil and to determine its potential for reducing toxic heavy metals. Cadmium reduction by microorganisms has suggested the importance of microorganisms in bioremediation and envi-ronmental cleanup operations (Niu et al., 1993; Chen and Wilson, 1997; Morris et al., 1999). The tolerance of isolated cells to elevated Cd2+ in the environment and the biochemi-cal basis of this tolerance, as well as the metal adsorption capacity of the cells, were examined.
2. Methods
2.1. Isolation of heavy-metal-resistant microorganisms
YPG media (peptone, 10g; yeast extract, 5g; gluco, 10g) was sterilized by autoclaving at 120°C for 15min. A sterilized solution of 0.5M CdCl2 through a Millipore W lter was aptically added to the medium at a W nal concentra-tion of 2mM. Soil obtained from random areas was dis-solved in 10–20 volumes of sterilized physiological saline and was spread onto a YPG plate containing 2mM of Cd2+ and 50 g/ml of chloramphenicol. After incubation at 30°C for 3 days, colonies formed were removed and streaked onto another plate, and this was ud as the experimental strain.
2.2. Determination of chlorophyll
The growth at 30°C in YPG broth (pH 7.0) containing gluco was determined by measuring packed cell volume per liter of culture using a hematocrit and cell weight (dry weight of cells, g/l). Chlorophyll a and b levels were deter-mined by measuring the optical density at 660nm and 642.5nm in ethyl ether cell extract, and were calculated according to Je V rey’s equation (Je V rey, 1976). Cells were then obrved by X uorescence microscopy (E xciter: 520–550nm, Dichroic mirror: 565nm, Emitter: 580nm) (Olym-pus, Tokyo).
2.3. Saccharide utilization
The pre-culture was maintained in 3ml of yeast extract peptone dextro (YPG) broth (pH 7.0) at 30°C for 2 days in the dark. Assimilation of various carbon sources was investigated using MBM medium [KNO3, 25mg; MgSO4·7H2O, 7.5mg; K2HPO4, 7.5mg; KH2PO4, 17.5mg; NaCl, 2.5mg; CaCl2·2H2O, 1mg; Fe-mixture (FeSO4·7H2O, 1g; distilled water, 500ml), 0.1ml; A5-metal mixture (H3BO3, 286mg; MnSO4·7H2O, 250mg; ZnSO4·7H2O, 22.2mg; CuSO4·5H2O, 7.9mg; Na2MoO4, 2.1mg; distilled water 100 m1), 0.1ml; distilled water 99.8ml; pH 6.0] with or without 0.5% (w/v) of each saccha-ride. A total of 1ml of pre-cultured cells was inoculated into 100ml of MBA broth. MBA b
roth was then shaken at 150rpm at 30°C for 5 days. Growth was compared by mea-suring optical density at 550nm.
2.4. Optimum pH and temperature for growthisp是什么
The pre-culture was maintained in 3ml of YPG broth (pH 7.0) at 30°C for 2 days in the dark. The heterotrophic culture was performed using 100ml of YPG broth (pH 7.0) in each 300ml cotton plugged Erlenmeyer X ask by adding 1ml of the pre-culture broth, on a rotary shaker (100rpm) at 20–40°C for 4 days in the dark. The growth was com-pared by measuring the optical density at 550nm. The opti-mum pH for growth was determined using YPG broth that was adjusted to a desired pH value with 0.1N HCl or 0.1N NaOH.
2.5. Ampli W cation and quencing of 18S rDNA
上海世外小学学费Isolated microorganisms were grown in 3ml of YPG broth at 30°C for 3 days by agitation. DNA was extracted from 1.5ml of culture broth (approximately 0.04g cells) using a DNA extraction kit (Isoplant II; Nippon Gene, Toyama) according to the manufacturer’s instructions. Sen and antin primers were designed (5Ј-ACGGAGG ATTAGGGTTCGATTCCG-3Ј and 5Ј-GCTTCCATTG GCTAGTCGCCAATA-3Ј, respectively). Both primers were ud in PCR with chromosomal DNA acti
werkng as a tem-plate. The reaction mixture contained 5 g of chromosomal DNA, 400pmol of each oligonucleotide primer, and 0.1U of Taq DNA polymera in a volume of 50 l. Thirty ther-mal cycles of 94°C for 1min, 50°C for 1min, and 72°C for 1min, were carried out. PCR products were puri W ed by aga-ro gel electrophoresis. The resulting DNA fragment (1800 bp) was ligated into a pCRII vector (Invitrogen, CA, USA). The PCR-ampli W ed fragment was quenced using the dideoxy chain termination method (Sanger et al., 1977). Searches for similar quences were carried out using the BLAST program (Altschul Gapped BLAST 2001). Multi-ple alignments were run using GENETIX ver. 10. Distance matrix trees were constructed using Kimura’s two-para-meter model (Kimura, 1980).
2.6. Metal resistance and minimum inhibitory concentrations
Resistance of the isolated strain to cadmium chloride, cupric sulfate, zinc chloride, nickel chloride and aluminum chloride was determined by the dilution method (Nieto et al., 1989). Metal ions were added parately to YPG broth at concentrations of 1–10mM. YPG broth without the metal ions was also inoculated with the isolated strain for u as controls. Each broth was cultured at 30°C for 4 days by agitation at 100rpm. The minimum inhibitory con-centration (MIC) was de W ned as the lowest concentration of metal that completely inhibited growth.
学士学位翻译
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2.7. Uptake of heavy metals by isolated microorganism
The isolated microorganism was cultured in YPG broth at 30°C for 4days and was then harvested by centrifugation at 2000g for 15min. Next, 100mg (dry weight) of harvested cells were suspended in 100ml of 10mM Tris–HCl bu V er (pH 7.0) containing 1–5mg of either Cd2+, Zn2+ or Cu2+, or 2.5mg each of Cd2++Zn2+, Cu2++Zn2+, and Cd2++Cu2+. The suspended solutions were shaken at 100rpm at 30°C. Residual heavy metals in the upper pha following centri-fugation at 2000g for 15min were quanti W ed by atomic absorption spectrophotometry (Shimadzu, Japan).
mule2.8. Possibility of bioremediation
Rice eds (Oryza sative L. cv. Koshihikari) were surface sterilized in 70% (v/v) aqueous ethanol for 15min, rind 5 times with distilled water and allowed to germinate on a sheet of moist W lter paper at 25°C with a 12-h photoperiod in a growth chamber. Light was provided from above with a white X uorescent tube. After 3days, uniform edlings (n D30) were transferred onto a sheet of plastic mesh (3£3cm) that was X oated on distilled water (100ml) in the prence or abnce of 5mg/l of Cd2+ in plastic containers (5£5£8cm). Next, 0.03–0.50g (wet weight) of the isolated microorganism was adde
d to the media. Seedlings were grown at 25°C with a 12-h photoperiod. The water in the plastic container was kept at the same level by adding dis-tilled water at 24-h intervals and only the roots of the ed-lings were immerd in the water during the incubation. After 3 weeks, the shoot length was measured.
3. Results
3.1. Isolation of heavy-metal-resistant microorganisms
Soil samples were collected from various districts in Japan and more than a 100 strains were found to be Cd2+ and chloramphenicol resistant. A yeast-like strain that formed large green colonies was lected and its physiologi-cal pro W le was subquently examined. This strain, desig-nated ANA9, was then ud as the experimental organism. The cells of the isolated microorganism were always spheri-cal and were 5.35–8.56 m in diameter. The cells were grown on YPG and were always green, regardless of light-ing conditions.
3.2. Determination of chlorophyll
Strain ANA9 was obrved as bright red cells on X uores-cence microscopy, thus indicating that it po
ssd chloro-plasts. Chloroplasts were excited at 520/550nm and emission signals were collected at wavelengths above 580nm. The heterotrophic cells contained chlorophylls a and b (96 and 34 g/mg dry weight, respectively) and carotenoids (2.2 g/mg dry weight). Chlorophylls were con-stituents of chloroplasts, but were synthesized even under dark conditions when cells grew by metabolizing an organic carbon source.
3.3. Saccharide utilization
Utilization of organic carbon sources was investigated on MBM medium containing 0.5% of each sample in the dark. Only a few were found to support growth as the sole carbon source: gluco, sucro, malto, galacto and sol-uble starch. Cellobio, sorbo, ra Y no, xylo and lac-to were unable to support growth under the experimental conditions ud. Most algal strains showed higher growth rates under autotrophic conditions than under heterotro-phic conditions. In contrast to the strains, heterotrophic growth rate of strain ANA9 was rather higher than the autotrophic rate. Chlorella ellipsoidea(Yamada and Shi-maji, 1986) was ud as a control and was unable to grow under heterotrophic or dark conditions.
3.4. Optimum pH and temperature for growth
The optimal temperature and pH for growth under het-erotrophic conditions without light were found to be 35°C and pH 6.0–7.0, respectively (Fig.1). Isolated strain ANA9 did not exhibit substantial growth at pH>8.0 or <4.0. The growth at 40°C was suppresd, thus suggesting that this was not a thermophilic microorganism.
1846N. Yoshida et al. / Bioresource Technology 97 (2006) 1843–1849
3.5. Heterotrophic growth
The cells were cultured on YPG medium in which gluco concentration was adjusted 0–4% (w/v). As shown in Fig.2, the optimum concentration of gluco for growth was 3%, where the cell yield was 50g/l after culturing at 30°C for 120h. The growth rate and yield of a number of micro-algae have been reported (Endo et al., 1974). Compared with the strains, isolated strain ANA9 can be recognized to have a much higher growth under heterotrophic conditions.
3.6. Ampli W cation and quencing of 18S rDNA
The small subunit ribosomal RNA gene of strain ANA9 was ampli W ed from bulk genomic DNA by PCR. The iso-lated small subunit rRNA quence was 1870 nucleotides long and showed similarities with other known quences from green algae, ranging in homology from 98% with Chlorella sorokiniana (Huss et al., 1999) to 95% with Chlo-rella saccharophila (Krienitz et al., 1996). Strain ANA9 was thus identi W ed as belonging to the genus Chlorella. Strain ANA9 was subquently identi W ed as belonging to the spe-cies C. sorokiniana and was therefore designated C. soroki-niana ANA9.
3.7. Metal resistance and minimum inhibitory concentrations
The MICs of 5 metal ions tested against strain ANA9 are shown. For the purpo of de W ning metal resistance, strains that were not inhibited by 1mM of heavy metal ions were regarded as resistant (Nieto et al., 1989). The isolate was resistant to multiple metals. The isolated microorgan-ism was particularly resistant to the W ve metal ions, the highest MIC was en for Cd2+ (4mM). Strain ANA9 was able to grow in YPG broth containing Cd2+ at concentra-tions of 3mM. This strain had a higher resistance to Cu2+ and Al3+ (MICs of 8mM and 7mM, respectively) than to Cd2+, Zn2+ and Ni2+ (MICs of 4mM). In the prent study, toxicity of metal ions was ranked as follows: Cd2+D Zn2+D Ni2+>Al3+>Cu2+.
3.8. Uptake of heavy metals by the isolated microorganism
Fig.3 shows the time cour of heavy metal uptake by the isolated microorganism. The amount of Cd2+, Zn2+ and Cu2+ taken up by the cells incread rapidly during the W rst 30min after the application of cells, and then steadily incread with time. When the initial amount of Cd2+ was 5mg, 55% was taken up from the Tris–HCl bu V er after 24h
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N. Yoshida et al. / Bioresource Technology 97 (2006) 1843–18491847
and 90% was taken up after 48h. When the initial amount of Cd 2+ was 2.5 and 1mg, 90% of the Cd 2+ was also taken up after 48h. When the initial amount of Zn 2+ was 5mg, the reduction in Zn 2+ concentration was 75% after 24h and 85% after 48h. The adsorption of Cu 2+ by the isolated strain was W rst very rapid within the W rst 30min, resulting in more than 80% being taken up. The maximum amounts of Cd 2+, Zn 2+, and Cu 2+ taken up by the cells were 43.0,42.0, and 46.4 g, respectively, per milligram of dry weight.The e V ects of the prence of co-ions in the solution on the heavy metal uptake capacity of the isolated organism were also examined. As en in Fig.4, Zn 2+ had no e V ect on Cd 2+ uptake, despite the 2.5mg co-ion concentration. The Cd 2+ and Zn 2+ uptake capacity of the isolated strain was
progressively and negatively a V ected by Cu 2+, indicating that the isolated strain had a higher a Y nity for Cu 2+ than for Cd 2+ and Zn 2+. After 48h of incubation, 50% and 80%of the Zn 2+ and Cu 2+, respectively, was removed from the solution. After 48h incubation, 60% and 80% of the removed Cd 2+ and Cu 2+, respectively, was accumulated intracellularly or adsorbed on the cell surface. The isolate exhibited a higher lectivity for Cu 2+ than for other the metal ions and the preference was as follows: Cu 2+>Cd 2+>Zn 2+.
The distribution and binding states of Cd 2+ in the isolated microorganism were examined by washing with 10mM EDTA solution. Isolated microorganism cells were suspended in 10mM EDTA solution (pH 5.0) by agitation at room temperature after incubation with Cd 2+ for 48h.After 5min in the EDTA solution, the supernatant was col-lected by centrifugation at 2000g for 20min. None of Cd 2+taken up in the living cells was relead by washing with E DTA. This indicates that the cells incorporated Cd 2+intracellularly.
3.9. Potential for bioremediation
The e V ectiveness in reversing Cd 2+-inhibited plant growth was determined. At concentrations of 5mg/l, Cd 2+inhibited hypocotyl and shoot growth of rice edlings. The shoot growth of rice edlin
gs was restricted to 60% in the prence of Cd 2+ when compared with control rice ed-lings. The shoot growth of rice inhibited by Cd 2+ incread with Chlorella cell concentration. As little as 0.25g (wet weight) of Chlorella cells prevented Cd 2+ toxicity. Smaller amounts, 0.03–0.1g (wet weight), of Chlorella cells had no e V ect on the growth inhibition by Cd 2+ (Fig.5). The results suggest that the isolated Chlorella cells had the potential for bioremediation of rice husks by preventing Cd 2+ accumulation near the plants.4. Discussion
Cadmium was an important environmental pollutant and a potent toxicant to bacteria, algae and fungi. Mechanisms
Fig.5. E V ects of Cd 2+ on rice shoots grown under hydroponic conditions with or without isolated strain ANA9. Vertical bars reprent SE (n D 30).
1848N. Yoshida et al. / Bioresource Technology 97 (2006) 1843–1849
of Cd2+ toxicity and resistance varied depending on the
organism. However, the form of the metal and the environ-
ment it is studied in play an important role in how Cd2+ exerted its e V ects and how organisms resp
ond. A wide
托福培训学校range of Cd2+ concentrations have been ud to designate
resistance in organisms. To date, however, no particular
concentration has been speci W ed as applicable to all species under standardized conditions. Cadmium exerted its toxic
e V ects over a wide range o
f concentrations. In most cas,
algae and cyanobacteria were the most nsitive organisms,
while bacteria and fungi appear to be more resistant. In some bacteria, plasmid-encoded resistance could lead to
reduced Cd2+ uptake, although some Gram-negative bac-
teria without plasmids were just as resistant as bacteria
equity
containing plasmids encoding Cd2+ resistance.
Insu Y cient information is available on the genetics of
Cd2+ uptake and resistance in cyanobacteria and algae, and
mechanisms remain largely unknown. Cd2+ was toxic to
the organisms, causing vere inhibition of physiological process, such as growth, photosynthesis and nitrogen W xation, at concentrations of less than 2ppm, and often in the ppb range (Perfus-Barbeoch et al., 2002). Cd2+ also
caud pronounced morphological aberrations in the organisms, and the were probably related to deleterious e V ects on cell division. Such e V ects may be direct or indi-rect, perhaps as a result of Cd2+ e V ects on protein synthesis and cellular organelles such as mitochondria and chloro-plasts. Cd2+ accumulated internally in algae (Perez-Rama et al., 2002) as a result of a two-pha uptake process. The W rst pha involved rapid physicochemical adsorption of Cd2+ onto cell wall binding sites, which were probably pro-teins and/or polysaccharides. This was followed by a lag period and a steady intracellular uptake. This latter pha was energy dependent and may involve th
e transport sys-tems ud to accumulate other divalent cations, such as Mn2+ and Ca2+. Some data indicated that Cd2+ resistance and possibly uptake in algae and cyanobacteria were controlled by plasmid-encoded genes (Ren et al., 1998). Although considerable information is available on Cd2+toxicity to and uptake in fungi, further work was clearly needed in veral areas. There was little information regarding Cd2+ uptake by microalgae, W lamentous fungi, or even in yeast, while information on speci W city, kinetics, and mechanisms of Cd2+ uptake was limited.
Chlorella sp. is a unicellular green alga having worldwide
distribution in all aquatic environments, soil (Koelewijn
et al., 2001) and tree bark (Cho et al., 2002). It is also found as a symbiote in animals such as Hydra viridis(Habetha and Bosch, 2005). Chlorella only reproduces axually, with the mature cell dividing mitotically to produce autospores. Chlorella is a very small alga with a single chloroplast, and it does not posss an eyespot or X agella. Algae of genus Chlorella can grow photosynthetically, and certain strains among the are known to grow in the dark by utilizing organic carbon sources (Rehman and Shakoori, 2001). It has been reported that the growth rate of algae was gener-ally much slower than bacteria and yeast, and dark hetero-trophic growth was even slower. However, if s
trains of algae could be found that posss higher growth rates under heterotrophic conditions, then production of algal proteins by industrial means may become more practical.
C. sorokiniana ANA9, which had a much higher growth rate under heterotrophic conditions than other reported strains, was successfully isolated from soil. This isolated alga possd a high growth rate (50g/l) in batch culture.
The heavy metal binding potential of Chlorella sp. was W rst discovered in the mining industry, and metal-resistant algae have since been reported in a number of studies. Matsunaga et al. (1999) screened marine microalgae for e Y cient Cd2+ removal with the aim of onsite heavy metal removal from the marine environment. They reported a maximum uptake in Chlorella of 39.4 g Cd2+/mg dry cells, which was higher than that of any other previously charac-terized microalgae. The maximum amounts of Cd2+, Zn2+, and Cu2+ taken up by the prent isolate were 43.0, 42.0, and 46.4 g, respectively, per milligram (dry weight). Thus, the isolated C. sorokiniana ANA9 had the highest metal adsorption ever reported for algae.
Agricultural soils were primarily contaminated with Cd2+ due to the excessive u of phosphate fertilizers, dis-persal of wage sludge and atmospheric deposition. Cd2+ was readily taken up by n
umerous crops including cereals, potatoes, rice and fruits (Ingwern and Streck, 2005). Con-sumption of rice grown in paddy soils contaminated with Cd2+, Cr6+ or Zn2+ may po a rious risk to human health, becau 22–24% of the total metal content in rice biomass was concentrated in the rice grains (Wang et al., 2003). Thus, contamination by Cd2+ is increasing in both human food and overall in the agricultural environment. Plants readily take up Cd2+ from the soil. However, expo-sure to high levels of Cd2+ resulted in reduced rates of pho-tosynthesis, chlorosis, growth inhibition, browning of root tips, decread water and nutrient uptake, and ultimately death (Marcano et al., 2002). Phytoremediation technolo-gies are becoming recognized as cost-e V ective methods for remediating sites contaminated with toxic metals at a frac-tion of the cost of conventional technologies, which include soil replacement, solidi W cation and washing strategies. Phy-toremediation is de W ned as the u of plants for environ-mental cleanup, and in terms of phytoremediation of heavy metals, is divided into three categories; (1) phytoextraction, in which metal-accumulating plants are ud to transport and concentrate metals from the soil in harvestable parts of roots and above-ground shoots; (2) rhizo W ltration, in which plant roots absorb, precipitate and concentrate toxic metals from polluted e Z uents; and (3) phytostabilization, in which heavy-metal-tolerant plants are ud to reduce the mobility of heavy metals, thereby reducing the risk of further environmental degradation by leaching into ground water or by airborne spread. Here, we ide
nti W ed the potential for metal-accumulating Chlorella to reduce the mobility of heavy metals in soil. As shown in Fig.5, our results indicated that the isolated C. sorokiniana ANA9 may be uful in preventing Cd2+ di V usion in the soil environment.
