Genetic diversity of Kappaphycus species (Gigartinales,Rhodophyta) in the Philippines

The mariculture of eucheumatoids (species of Kappaphycus and Eucheuma) in the Philippines has had a long history, dating back to the 1970s. Over this period, a number of varieties have been brought into domestication; some are now widely distributed and farmed in various regions of the country, but a significant number appear to have a more restricted distribution and are farmed only in certain areas. The taxonomy of many of these seaweed cultivars and their phylogenetic relationships still remain to be resolved at the specific and subspecific levels. In this study, two mitochondrial DNA markers, COI-5P region and cox2-3 intergenic spacer, were used to assess the genetic diversity of the farmed varieties and a few specimens collected from the wild. Analysis using haplotype networks revealed several new haplotypes for K. alvarezii, K. malesianus and K. striatus, mainly from specimens collected from eastern and southwestern regions of the Philippines. The inferred phylogenetic relationships based on both mtDNA markers resolved the identity of all the materials used in the study at the species level. We present molecular evidence that K. malesianus, in addition to K. alvarezii, K. cottonii, K. inermis, K. procrusteanus and K. striatus (and, hence, all currently recognized species of Kappaphycus) occurs in the Philippines. Collectively, these observations suggest that the Philippine archipelago has richer genetic diversity of farmed and wild Kappaphycus than do the other geographic regions, consistent with the hypothesis that the Philippines is, or is part of, the centre of Kappaphycus biodiversity in the world. These findings also reveal an untapped diversity that can potentially be exploited for improving the commercial production of these carrageenophytes.     

Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

Systematics and genetic variation in commercial shape Kappaphycus and shape Eucheuma (Solieriaceae, Rhodophyta)

The systematics and taxonomy of Kappaphycus and Eucheuma (Solieriaceae) is confused and difficult due to morphological plasticity, lack of adequate characters to identify species and commercial names of convenience. These taxa are geographically widely dispersed through cultivation. Commercial, wild and herbarium sources were analysed; molecular markers provided insights into taxonomy and genetic variation, and where sources of genetic variation may be located. The mitochondrial cox2-3 and plastidal RuBisCo spacers were sequenced. There is a clear genetic distinction between K. alvarezii (“cottonii”) and K. striatum (“sacol”) samples. Kappaphycus alvarezii from Hawaii and some samples from Africa are also genetically distinct. Our data also show that all currently cultivated K. alvarezii from all over the world have a similar mitochondrial haplotype. Within Eucheuma denticulatum (“spinosum”) most African samples are again genetically distinct. Our data also suggest that currently cultivated E. denticulatum may have been “domesticated” several times, whereas this is not evident for the cultivated K. alvarezii. The present markers used do not distinguish all the morpho-types known in cultivation (e.g. var. tambalang, “giant” type) but do suggest that these markers may be useful to assess introductions and species identification in samples.     

Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

A comparative analysis of phycocolloids produced by underutilized versus industrially utilized carrageenophytes (Gigartinales, Rhodophyta)

Carrageenan (E-407) and semi-refined carrageenan (E-407a) are some of the main additives used by the food industry for their gelling, emulsifying, thickening, and stabilizing properties. These are natural ingredients, which have been used for decades in food applications and are generally regarded as safe. Internationally, sub-tropical carrageenophytes (e.g., Kappaphycus alvarezii) are cultivated extensively as a source of raw materials for industrial extraction, and their use as potential candidates in integrated multitrophic aquaculture is tentative. We analyzed carrageenan yield (as a percentage of dry weight) and chemical composition (by Fourier transform infrared attenuated total reflection and Fourier transform-Raman) of extracts produced by several carrageenophytes (Gigartinales, Rhodophyta), from different origins, e.g., K. alvarezii (Tanzania, Indonesia, the Philippines, Panama, and Mexico), Kappaphycus striatum (Madagascar), Eucheuma denticulatum (Tanzania, the Philippines, and Madagascar), Betaphycus gelatinum (the Philippines), Chondracanthus chamissoi, and Sarcothalia crispata (Chile). For comparison, some underutilized carrageenophytes were also analyzed, e.g., Chondrus crispus, Mastocarpus stellatus, Gigartina pistillata, Chondracanthus teedei var. lusitanicus, Chondracanthus acicularis, Calliblepharis jubata, Gymnogongrus crenulatus, and Ahnfeltiopsis devoniensis (Portuguese carrageenophytes). The main findings were that the highest carrageenan yield was obtained from K. striatum (Madagascar) with 75.6 (percent dry weight (% DW)); B. gelatinum and K. alvarezii (both from the Philippines) had yields of 71.0% and 68.0% (% DW), respectively; and G. pistillata (Portugal) 65.4% (% DW). Spectroscopic analysis of the phycocolloids allowed determination of a wide range of carrageenan types, e.g., pure iota carrageenan, several kappa–iota hybrid carrageenans with different iota/kappa ratios, and kappa–beta, xi–theta, and xi–lambda hybrid carrageenans.     

Investigation of the application of Acadian Marine Plant Extract Powder (AMPEP) to enhance the growth, phenolic content, free radical scavenging, and iron chelating activities of Kappaphycus Doty (Solieriaceae, Gigartinales, Rhodophyta)

An optimization study on concentration (viz. 0.01, 0.1, and 1.0 g L^?1) and dipping time (i.e., 30 and 60 min) was conducted on three different color morphotypes (i.e., reddish brown, yellowish brown and purple) of the commercial carrageenophyte Kappaphycus alvarezii (Doty) Doty. The study tested the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth rate and occurrence of macro-epiphytes from August to November, representing the wet season of the Philippines. The optimum concentration and dipping time were obtained at 0.1 g L^?1 and 30 min, respectively. These optimum parameters were then further verified in a commercial nursery using the yellowish brown morphotype. In another experiment, K. alvarezii (tambalang purple morphotype) and Kappaphycus striatum (Schmitz) Doty (sacol green morphotype) with, and without, AMPEP dippings were tested for their total phenolic content, free radical scavenging and iron chelating activities. Seaweed dipped in AMPEP demonstrated higher growth rates than the control. Lower concentrations (i.e., 0.01-0.1 g L^?1) and shorter dipping time (e.g., 30 min) produced higher growth rates than the highest concentration (1.0 g L^?1) and longer (60 min) dipping time. The presence of macro-epiphytes such as filamentous Ulva did not adversely affect the robust growth of the three color morphotypes of K. alvarezii. The lowest and highest growth rates obtained in a commercial seaweed nursery using the optimum concentration and dipping time of AMPEP were observed in July and January with 0.8% and 6.7% day^?1, respectively. The antioxidant content of K. alvarezii (tambalang purple) and K. striatum (sacol green) responded differently to AMPEP dipping. The changes in total antioxidant activity followed almost the same trend as in phenolic content, in both K. alvarezii (tambalang purple) and K. striatum (sacol green), whereas, the iron chelating ability of both seaweeds with and without AMPEP dipping varied monthly. The results obtained for the use of AMPEP dips for commercial Kappaphycus cultivation demonstrated an effective management tool for improved farming protocols.     

Introduction of non-native species of Kappaphycus (Rhodophyta, Gigartinales) in subtropical waters: Comparative analysis of growth rates of Kappaphycus alvarezii and Kappaphycus striatum in vitro and in the sea in south-eastern Brazil

We compared the growth rates of Kappaphycus alvarezii (Doty) Doty ex P. Silva and Kappaphycus striatum (Schmitz) Doty, both, in vitro under different conditions of light and temperature, and in the sea. Temperature was the most important factor controlling the growth of both species, in vitro and in the field. In the sea there was a clear seasonal pattern in growth rate, attributed to temperature and salinity variation. The lower growth rates were registered in winter and spring, and the highest in summer and autumn months for both species. Based on growth rate in the field, and the production of viable tetraspores during the summer in Kappaphycus striatum, we conclude that is more profitable, and ecologically safer, to only continue with the introduction program of Kappaphycus alvarezii.     

Impact of AMPEP on the growth and occurrence of epiphytic Neosiphonia infestation on two varieties of commercially cultivated Kappaphycus alvarezii grown at different depths in the Philippines

Two varieties of the carrageenophyte Kappaphycus alvarezii (Tungawan, TUNG; and Giant tambalang, GTAM) from Zamboanga Sibugay, Philippines were used to test the efficacy of Acadian Marine Plant Extract Powder (AMPEP) as source of nutrients for growth, and to determine if applications had any effect on the percent occurrence of an epiphytic infestation of the red alga Neosiphonia sp. at four different depths in the sea. Results showed that the use of AMPEP significantly (P < 0.05) increased the growth rate of both Kappaphycus varieties tested but decreased the percent occurrence of Neosiphonia sp. The percent occurrence of Neosiphonia sp. infection (6–50% at all depths) of both Kappaphycus varieties with AMPEP treatment was significantly lower than the controls (i.e., 10–75% at all depths). Both the growth rate of the cultivated seaweed and the percent occurrence of the epiphytes decreased as the cultivation depth increased. Plants dipped in AMPEP and suspended at the surface had the highest growth rates (i.e., 4.1%, TUNG; 3.1%, GTAM) after 45 days; those without AMPEP dipping had the highest percent occurrence of Neosiphonia infection (viz. 70–75%). The occurrence of Neosiphonia infestation was found to be correlated with changes in irradiance and salinity at the depths observed. The results suggested that both varieties of K. alvarezii used in this study have the fastest growth rate when grown immediately at the water surface. However, in order to minimize damage caused by the occurrence of epiphytic Neosiphonia, K. alvarezii should be grown within a depth range of 50–100 cm. These observations are important for the improved management of Kappaphycus for commercial farming. Furthermore, the use of AMPEP treatments for enhancement of growth and reduction deleterious Neosiphonia sp. infections is encouraging.     

Cultivation and carrageenan yield and quality ofKappaphycus alvarezii in the waters of Vietnam

The carrageenan-producing red algaKappaphycus alvarezii (Doty) Doty was brought to Vietnam from Japan in 1993. Branch fragments of this species were cultivated in a pond, lagoon, inlet and offshore in Vietnam for the first time. The best daily growth rate (DGR) of plants grown in the lagoon area attained 9–11 % day^–1 in May to June (cold season). The water temperature and salinity in this area ranged from 27.2–32.4 °C and 31.4–33.7 °C, respectively. DGR of plants grown in the inlet ranged from 7 to 9% day^–1 in June. Grazing by fish has been observed to occur in this area. The DGR of plants grown in the pond ranged from 5–6% in January–July, but decreased to less than 4% day^–1 in August (hot season). K. alvarezii in Vietnam showed a carrageenan yield of 18.8–24.6% and gel strength of 1566–1712 g cm^–2. These values are similar ones obtained fromK. alvarezii cultivated in the Philippines and Indonesia.     

A molecular method for identification of the morphologically plastic invasive algal genera Eucheuma and Kappaphycus (Rhodophyta, Gigartinales) in Hawaii

A paucity of diagnostic morphological characters for identification and high morphological plasticity within the genera Eucheuma and Kappaphycus has led to confusion about the distributions and spread of three introduced eucheumoid species in Hawaii. Entities previously identified as E. denticulatum, K. alvarezii, and K. striatum have had profound negative effects on Oahu’s coral reef ecosystems. The use of molecular tools to aid identification of algal species has been promising in other morphologically challenging taxa. We used three molecular markers (partial nuclear 28S rRNA, partial plastid 23S rRNA, and mitochondrial 5′ COI) and followed a DNA barcoding-like approach to identify Eucheuma and Kappaphycus samples from Hawaii. Neighbor-joining analyses were congruent in their separation of Eucheuma and Kappaphycus, and the resulting clusters were consistent with those revealed for global comparisons with the mitochondrial cox2-3 spacer and GenBank data. Based on these results, new insights were revealed into the distribution of these groups in Hawaii.     

Karyology,nuclear genome quantification and characterization of the carrageenophytesEucheuma andKappaphycus (Gigartinales)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteKappaphycus alvarezii. Results indicate the presence of three second order components corresponding to fast (12%), intermediate (38%) and slow (50%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Comparison of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in pg/2C genome estimates:Eucheuma denticulatum=0.35,E. isiforme=0.44,Kappaphycus alvarezii=0.32 andK. striatum=0.42. Karyological studies of tetraspore mother cells during diakinesis using aceto-orcein revealed a chromosome complement of 10 forEucheuma denticulatum andKappaphycus alvarezii.     

Recruitment of Eucheuma and Kappaphycus on a farm in Tawi-Tawi,Philippines

Vegetative regeneration is the only farming method for Eucheuma and Kappaphycus, the world's most important carrageenophytes. Lack of seedstock has been a problem following natural calamities, overharvesting, grazing and outbreak of "ice-ice" disease. Hence, studies to help develop an alternative method of generating Eucheuma and Kappaphycus sporelings on a seaweed farm in Tawi-Tawi, Philippines were carried out from May 1993 to July 1994. Mactan stone blocks were deployed in two positions at two sites in Tawi-Tawi, one of the biggest farming areas in the Philippines. Recruits were first observed five months after deployment. Nested ANOVA was used to investigate the effect of current speed (C), water movement (M), number of days with minus tide (D), salinity (S), temperature (T), area of deployment (A) and position of blocks (P) on the recruitment of sporelings (R). Since C, M, D, S, and T were always on the same mean level within an area, the effects of these variables were assumed by the effect of area and thus, the A and P variables were the ones fitted in the ANOVA model. Repeated measures ANOVA showed no significant difference in the effects of A and P on visible R from May to November 1993 and March to July 1994. The Helmert transformation showed that mean recruitment of Eucheuma and Kappaphycus between May 1993 and subsequent time points was significantly different. This was also true for recruitment in September 1993, December 1993, January 1994 and February 1994, which were highly significantly different compared to the means of subsequent time points. Multiple regression analysis showed that C, D, and S had significant effects on recruitment of Eucheuma and Kappaphycus in both areas while effect of T was significant only in area I.     

Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.     

Effect of ammonium on growth and carrageenan content in Kappaphycus alvarezii (Gigartinales,Rhodophyta)

Kappaphycus alvarezii, cultured in a region deficient in nitrogen with intermittent application of 10 mM ammonium at three-day intervals for 1 hour, attained a daily growth rate of 4.6%, which was considered to be the optimum growth rate. Under such conditions, the C/N ratio was 29 with a carrageenan content of 58% and gel strength of 45–70 g cm^–2. Gel strength of carrageenan was high when nitrogen content was high and vice versa.     

Laboratory-induced development of the ice-ice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The most common perception of unfavorable environmental factors causing the ice-ice disease in the farmed seaweeds, Kappaphycus and Eucheuma, was demonstrated in this study for the first time using stressful conditions of abiotic factors in a continuous culture system. Light intensity of less than 50 mol photon m^–2 s^–1 and salinity of 20% or less induced ice-ice whitening characterized by short segments at midbranches which were similar to those observed in the Philippine seaweed farms, while temperatures of up to 33–35 °C resulted in wide-scale whitening leading to complete damage of the branches. These effects were preceded by slow growth rates from an optimum of 3.7% d^–1 to almost –2.0% d^–1. Mechanical stress by wound injury did not result to ice-ice whitening similar to the above. Environmental factors observed to trigger ice-ice in the laboratory, although may not necessarily parallel those in the field, may act synergestically to produce similar effects.     

Factors Influencing the Growth Rates of Three Commercial Eucheumoids at Coastal Sites in Southern Kenya

As a possible means of improving the livelihoods of local villagers, off-bottom rope cultivation of commercial eucheumoids was investigated on the southern Kenyan coast at three sites, representative of the variety of environments. The morphotypes used were brown Eucheuma denticulatum and green and brown Kappaphycus alvarezii. The study was carried out over a 15 month period from August 2001 until October 2002. Relative growth rates were highest at a sandy flat in a mangrove system (Gazi; 5.6% d⁻¹), and lowest in an intertidal reef flat (Kibuyuni; 3.2% d⁻¹) with a lagoon being intermediate (Mkwiro; 4.8% d⁻¹). The brown E. denticulatum had the highest growth rate of 4.7% d⁻¹ compared to the green and brown K. alvarezii which were 4.3% d⁻¹ and 4.2% d⁻¹, respectively. Growth was more variable at Kibuyuni and Mkwiro. The growth was higher during the southeast monsoon (4.7% d⁻¹) than during the northeast monsoon (4.0% d⁻¹). This is part of a larger study and the effects of water motion, salinity, temperature, thallus nitrogen, and ‘ice-ice’ syndrome on growth of morphotypes is discussed. The water motion was observed to increase thallus nitrogen and hence the growth of eucheumoids. The ‘ice-ice’ condition affected both brown E. denticulatum and brown K. alvarezii but not green K. alvarezii. The results suggest that commercial cultivation of eucheumoids in Kenya will be feasible.     

Cage culture ofKappaphycus alvarezii var.tambalang (Gigartinales,Rhodophyceae)

Kappaphycus alvarezii var.tambalang was cultured in a 3 × 3 m bamboo raft installed inside a 4 × 4 m floating net cage ofLates calcarifer (sea bass) broodstock at SEAFDEC Sub-station Igang, Guimaras, Philippines, from December 1989 to May 1990. Growth and production ofK. alvarezii var.tambalang was influenced by the culture months. The highest growth rate and production were recorded in January and May, respectively, while the lowest growth rate and production were observed in March. Average growth rate (wet weight) ranged from 3.72 to 7.17% day^-1, while production ranged from 575.5 to 2377 g m^-1 line^-1. A total production of approximately 123 t (fresh) or 37 t (dried) ha^-1 in the 5-month harvest season was produced from this culture system. Cultivation ofK. alvarezii var.tambalang in cages is possible, which indicates that seaweeds can be grown with carnivorous finfish, a practice which is still untapped.     

Laboratory and field growth studies of commercial strains ofEucheuma denticulatum andKappaphycus alvarezii in the Philippines

Daily growth rates of 0.1 to 8.4% d^-1 for the brown form and 0.2 to 6.3% d^-1 of the green form were measured for 3 to 5-cm long branches of the tropical red seaweedKappaphycus alvarezii cultured in the laboratory. Highest growth rates were found using inexpensive enrichments such as soil water and coconut water supplemented with 0.7 mM N and 13 µM P and with a liquid fertilizer, Algafer, produced from seaweeds in the Philippines. Laboratory grown branches of bothK. alvarezii andEucheuma denticulatum transplanted to rafts in the field showed daily growth rates of 4.4 to 8.9% d^-1, as high or higher than other reported growth rates. The studies, carried out in the Philippines, demonstrate the viability and high yield of laboratory cultivars and methods to keep laboratory culture costs low.     

Non-enzymatic isolation of somatic cells from Kappaphycus spp. and Eucheuma denticulatum (Solieriaceae,Rhodophyta)

Cell culture technology is immensely useful in somatic hybridization, induction of mutations, cloning of specific isolates and maintenance of strains of defined genotypes. However, its application in strain improvement of some tropical red macroalgae has been limited due to the difficulty of isolating viable cells from their complex intercellular matrices. A simple, non-enzymatic technique of isolating somatic cells was developed for Kappaphycus spp. and Echeuma denticulatum. Surface-sterilized tissues (0.1 g fresh weight, 2.0 mm thick discs) from subcortical and medullary layers were treated with 3% NaOH, 3% KOH, or hydrogen peroxide in phosphate buffer solution (PBH₂O₂). Tissues of K. alvarezii treated with PBH₂O₂ softened after 5 h of treatment and completely dissociated after 12 h. Viable cell counts (VCC), determined through staining with Evan’s blue, were significantly higher (2.4 × 10⁵ cells g^?1 fresh weight tissue) in K. alvarezii (‘tambalang’ strain) treated with PBH₂O₂ compared with tissues treated with carrageenase from a marine bacterium.     

Heavy metal chelation by non-living biomass of three color forms of Kappaphycus alvarezii (Doty) Doty

Water pollution by toxic heavy metals is a burning environmental problem and has presented a challenge to humans. Removal of heavy metals using non-living biomass of seaweeds could be a potential solution to this problem. In the present investigation, biomass of three color forms of Kappaphycus alvarezii, viz. brown, green and pale yellow, were studied in the laboratory for their heavy metal chelating capacity using cadmium, cobalt, chromium and copper. Amongst the four concentrations used (25, 50, 75 and 100 mg L⁻¹) maximum chelation of Cd, Co and Cu was recorded at 25 mg L⁻¹ concentration. The highest amount of Cr was chelated at 100 mg L⁻¹ by all the three color forms. The pale yellow form showed maximum chelation for all four metals studied. Further, chelation in all the color forms was found to be: Cd 5.37 ± 0.59–15.84 ± 0.32 %, Co 21.19 ± 0.13–32.32 ± 0.62 %, Cr 65.38 ± 0.27–88.09 ± 0.51 % and Cu 59.53 ± 0.37–90.28 ± 0.89 %. All the three color forms of K. alvarezii serve as an excellent biodetoxifier as they all chelated considerable amounts of heavy metals.