Genotyping‐by‐sequencing for construction of a new genetic linkage map and QTL analysis of growth‐related traits in Pacific bluefin tuna

Pacific bluefin tuna (Thunnus orientalis) has high market value, but its wild populations have decreased in recent years. The broodstock of Pacific bluefin tuna that were hatched artificially and reared under aquaculture conditions is beginning to be used for production. The creation of broodstock with commercially valuable traits, such as rapid growth, is therefore of great interest. Genetic linkage map‐based identification of markers associated with quantitative trait loci (QTLs) facilitates marker‐assisted selection (MAS) breeding and allows efficient genetic improvement of broodstock. Single nucleotide polymorphism (SNP)‐based genetic linkage map construction using the genotyping‐by‐sequencing method can expand the number of mapped markers and help identify growth‐related QTLs. In this study, we constructed sex‐specific maps for 24 linkage groups consisting of 677 SNP and 651 microsatellite markers. The total lengths of 93 progenies in the mapping population followed normal distribution, with an average length of 9.4 mm. We performed composite interval mapping in the mapping population. QTL analysis revealed one significant QTL in LG10 on the female linkage map. The genetic linkage map—the second such map generated for Pacific bluefin tuna—and the growth‐related QTLs detected in this study will be useful for tuna aquaculture MAS programs.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

Identification of quantitative trait loci for growth‐related traits in the swimming crab Portunus trituberculatus

The swimming crab (Portunus trituberculatus) is an economically important species in Asian aquaculture. Implementing growth‐related traits of P. trituberculatus into genetic breeding programmes is an ongoing effort. We used a previously published genetic linkage map of P. trituberculatus, containing 55 simple sequence repeat (SSR) markers and 172 amplified fragment length polymorphism (AFLP) techniques to map quantitative trait loci (QTL) for growth‐related traits in a single full sibling F₂ family. Ten growth‐related traits were measured for QTL mapping. Composite interval mapping identified 9 QTL on the female map and 16 on the male map. Individual QTL with additive effects explained 11–38% of the phenotypic variance for various traits using the female parent's map, and from 1% to 21% using the male parent's map. Two QTL explaining a large percentage of variation in body weight were detected on chromosome 17 on the female map, and on chromosome 16 on the male map, and contributed 38% and 18% of the phenotypic variance respectively. This is the first study to report the detection and positioning of major QTL affecting growth in a true crab species (Brachyura). The mapping of growth‐related QTL in this study raises the possibility of improving the growth of P. trituberculatus through marker‐assisted selection.     

长牡蛎出肉率与壳形性状的 QTL 定位分析

本研究以长牡蛎(Crassostrea gigas)F1全同胞家系为作图群体,在已构建的基于120个微卫星和66个SNP标记的长牡蛎性别平均连锁图谱上,利用PROC QTL 2.0软件对出肉率和壳形(壳宽和壳深)性状进行QTL定位分析。结果表明,共检测到13个相关的QTL,分布在3个连锁群上;其中,与出肉率相关的4个QTL定位在1号和3号连锁群上(LG1和LG3),表型解释率为0.25%~47.53%;与壳宽相关的3个QTL定位在10号连锁群上(LG10),表型解释率为0.71%~45.39%;与壳深相关的6个QTL也定位在LG10,表型解释率为3.37%~24.78%。根据QTL连锁群分析和性状相关性分析结果可以推测,出肉率与糖原含量性状以及壳宽与壳深性状分别具有相近的遗传特征,利用与相关性状共同关联的分子标记可以同时对出肉率与糖原含量性状、壳宽与壳深性状进行遗传改良。本研究结果为今后长牡蛎相关性状候选基因克隆和分子标记辅助育种提供了参考。     

Effects of Algae and Shell Pigment Extract‐supplemented Diets on Shell Pigmentation and Growth Performance of Pacific Abalone,Haliotis discus hannai

To improve shell pigmentation and growth performance of Pacific abalone, five extruded diets were prepared by supplementing a formulated control diet with 3% each of three species of algae (Pacific dulse, Porphyra yezoensis, Spirulina) and a pigment extract from the abalone shells (0.02%). Each of the five diets and a commercial feed were randomly assigned to three containers each stocked with 1000 juvenile Pacific abalone (2 g per individual) in a flow‐through seawater system for a 5‐mo feeding trial. Test results showed: (1) all the supplements significantly (P < 0.05) increased percentages of dark‐brown shelled abalone in the test treatments, relative to the control treatment; (2) the abalone fed the experimental diets achieved significantly (P < 0.05) greater final weight, shell‐length growth rates, and higher meat protein contents than those fed the commercial feed (P < 0.05); and (3) the abalone fed the Spirulina‐supplemented diet achieved the best overall growth, pigmentation performances, and feed conversion ratio; and the Pacific dulse supplement generated the highest protein content in abalone meat product (P < 0.05) among the tested diets. These results demonstrate the potential of locally made feed, which can generate desirable characteristics in abalone under aquaculture conditions.     

Suitability of cage culture for Pacific abalone Haliotis discus hannai Ino production in China

Pacific abalone (Haliotis discus hannai Ino) aquaculture is a thriving industry in China. This study describes a novel submerged cage culture system for abalone rearing in Fujian, South China. The cage consisted of five vertical slots that were oriented perpendicular to the flow of water. The slots were separated by six vertically connected plastic plates for abalone attachment and shelter at the bottom of the cage. Experiment 1 was designed to determine the appropriate stocking density at the start of the abalone sea‐based production cycle. Eight‐month‐old hatchery reared and size‐graded juveniles were transferred to the sea‐based culture system. For different stocking densities, shell length of juveniles obtained in this novel culture system on 2, 3.5 and 5 months, respectively, was compared with shell lengths obtained in a traditionally multi‐tier basket culture system. In Experiment 2, daily growth rates (DGRs) in shell length and biomass in terms of wet weight of 2‐year‐old abalones reared in cage and tiered basket culture systems were compared over a 6‐month period. Results of Experiment 1 showed that growth of abalone in the cage culture system is density‐dependent; the mean final shell length of juveniles obtained was 6.7–15.9% higher than in tiered baskets system even at the same initial stocking density. In Experiment 2, DGRs in shell length of 53.83–78.38 μm day^?1 obtained in cage system were significantly higher than that in tiered baskets (P < 0.01). And in terms of wet weight biomass, it was 1.48–3.01 times higher in the cage system compared with the traditional system. Abalone survival was more than 87.5% in both culture systems in both experiments. Advantages of the newly established cage culture system included better growth performance of the animals reared and potential improvement of rearing conditions, such as improved water flow velocity and dissolved oxygen.     

A genome scan of a four-way tilapia cross supports the existence of a quantitative trait locus for cold tolerance on linkage group 23

A genome scan, searching for quantitative trait loci (QTL) for the traits cold tolerance and body weight in tilapia, was performed on a cross between a (Oreochromis niloticus×Sarotherodon galilaeus) male and a (O. mossambicus×O. aureus) female. Fifty‐four microsatellites and 23 amplified fragment length polymorphism (AFLP) primer combinations were genotyped and tested for marker–trait associations. Sex‐specific linkage maps were constructed from this data. Twenty‐three point‐wise significant marker–trait associations were found in the genome scan, and putative QTL were subsequently tested in another (On×Sg) × (Om×Oa) family. None of the putative QTL from the first experiment were significant in the second experiment. However, one microsatellite, UNH130, found to be associated to weight in the first experiment, was found to be strongly associated to cold tolerance in the second experiment. Since QTL for cold tolerance and body weight were recently found on the linkage group containing UNH130 (linkage group 23) in another study, this linkage group was investigated more closely using interval mapping. The results provide indications, but not conclusive evidence, of a QTL for cold tolerance on linkage group 23.     

QTL mapping for glycogen content and shell pigmentation in the Pacific oyster Crassostrea gigas using microsatellites and SNPs

Glycogen content and shell pigmentation are two important economic traits of the Pacific oyster Crassostrea gigas. The first set of quantitative trait loci (QTLs) controlling the two traits was determined in an F1 full-sib family based on a sex-averaged linkage map. The linkage map was constructed using 120 SSRs, and 66 expressed sequence tag-derived single nucleotide polymorphisms (EST–SNPs). Two QTLs were found to be associated with glycogen content, explaining 0.27–79.05 % of the phenotypic variation. One QTL on LG9 were found to be related to shell pigmentation, the paternal and maternal alleles explaining 6.75 and 17.44 % of the phenotypic variation. The relationship of glycogen content with left shell depth and QTL linkage group analysis suggests that left shell depth and volume might be used to assist in indirect selection for glycogen content. The constructed linkage map and determined QTLs can provide a tool for further genetic analysis of the traits and be potential for marker-assisted selection in C. gigas breeding.     

Effects of diets on the growth performance and shell pigmentation of Pacific abalone

This study investigated the effects of natural and artificial diets on growth performance and shell pigmentation of cultured abalone. A 7‐month feeding trial was conducted on 12 000 Pacific abalones (Haliotis discus hannai) with four diets including two extruder‐processed test diets (with & without addition of oleopaprika carotenoids), Pacific dulse (Palmaria mollis) and a combination of Pacific dulse and a test diet. The results showed that the two test diets resulted in higher survival of abalone, but with a lower growth rates when compared with Pacific dulse alone (P < 0.05). The combination diet achieved the highest survival and growth rates. The Pacific dulse resulted in abalone with dark‐brown shells, which are preferred by Asian markets. The test diets led to 52% and 55% of the animals with pink coloured shells and the supplementation of oleopaprika did not affect shell pigmentation. The aqueous acidic extracts from both dark‐brown and pink shells showed blue colour; HPLC‐MS chromatography revealed that the pigments in the extracts were consistent with a biliverdin and a cysteine‐biliverdin. These results are valuable for the development of abalone feed and the control of abalone shell colouration.     

牙鲆遗传作图及生长性状QTL定位

采用牙鲆日本群体和韩国群体杂交的92个F₁个体作为分离群体, 利用微卫星标记和Joinmap 4.0作图软件构建了牙鲆遗传连锁图谱。共有221个SSR标记用于连锁图谱构建, 雌性图谱中, 共178个微卫星标记定位到22个连锁群上, 观测总长度为(G _oa )599.0 cM, 覆盖率(C _oa )达76.27%。雄性图谱中, 共194个微卫星标记定位到23个连锁群上, G _oa 为693.4 cM, C _oa 为78.82%。对全长、体质量、体高3组数据进行主成分分析处理, 得到可解释3个性状的89.6%特征的一组数据, 命名为牙鲆生长性状GT。用WinQTLCart 2.5软件的复合区间作图,在已构建的遗传连锁图谱上对牙鲆生长性状GT进行QTL定位, 取LOD经验值2.5为QTL存在的阈值; 对微卫星标记进行性状—标记之间的回归分析。本研究共定位3个与牙鲆生长性状GT相关的QTLs, qGT-f4 qGT-m20 qGT-f20,可解释表型变异率分别为27.60%, 13.74%, 10.27%。在性状—标记之间的回归分析中, 得到22个与生长性状GT相关(P<0.05)的微卫星标记, 单个标记可解释表型变异率介于3.70%~10.42%, 其中6个微卫星标记scaffold558_51720、scaffold558_26183、scaffold903_69232、scaffold485_47120 、scaffold1262_77386、scaffold809_65154与生长性状GT之间呈极显著相关(P><0.01), 可解释表型变异率分别为10.42%、7.31%、10.07%、10.07%、8.39%和11.26%。     

Heritability estimates for growth‐related traits in the Pacific oyster (Crassostrea gigas) using a molecular pedigree

Pacific oyster is one of the leading species in world aquaculture, but heritability estimation applying mixed‐family approach has not been actively pursued. In this study, heritability for growth‐related traits in the Pacific oyster was first estimated by creating a single cohort of 45 families in a full‐factorial mating design consisting of nine sires and five dams. A total of 270 offspring were analysed and parentage assignment inferred by six microsatellite markers achieved 100% success. All parents contributed to the spawn and a total of 42 full‐sib families were represented. Using an animal model, heritability estimates at 12 months of age were 0.49 ± 0.25 for shell height, 0.36 ± 0.19 for shell length, 0.45 ± 0.23 for shell width and 0.35 ± 0.17 for wet weight. Genetic correlation between shell height and wet weight was quite high (0.79 ± 0.25), suggesting that direct selection of shell height, which is an easily measurable trait, also improves wet weight. The results obtained in this study indicate that growth‐related traits could be improved by exploiting additive genetic effects through selective breeding.     

A first genetic linkage map of bluegill sunfish (<Emphasis Type="Italic">Lepomis macrochirus</Emphasis>) using AFLP markers

Genetic linkage maps were constructed for bluegill sunfish, Lepomis macrochirus, using AFLP in a F₁ inter-population hybrid family based on a double-pseudo testcross strategy. Sixty-four primer combinations produced 4,010 loci, of which 222 maternal loci and 216 paternal loci segregated at a 1:1 Mendelian ratio, respectively. The female and male framework maps consisted of 176 and 177 markers ordered into 31 and 33 genetic linkage groups, spanning 1628.2 and 1525.3 cM, with an average marker spacing of 10.71 and 10.59 cM, respectively. Genome coverage was estimated to be 69.5 and 69.3% for the female and male framework maps, respectively. On the maternal genetic linkage map, the maximum length and marker number of the linkage groups were 122.9 cM and 14, respectively. For the paternal map, the maximum length and marker number of the linkage groups were 345.3 cM and 19, respectively, which were much greater than those on the maternal genetic linkage map. The other genetic linkage map parameters of the paternal genetic linkage map were similar to those in the maternal genetic linkage map. For both the female and male maps, the number of linkage groups was greater than the haploid chromosome number of bluegill (2n = 48), indicating some linkage groups may distribute on the same chromosome. This genetic linkage mapping is the first step toward to the QTL mapping of traits important to cultured breeding in bluegill.     

An Assessment of Hatchery Cohort Growth Rates of South African Abalone,Haliotis midae,Across Four Commercial Environments

The abalone, Haliotis midae (commonly referred to as “perlemoen”), is an important aquaculture species in South Africa, accounting for the highest percentage of total aquaculture production revenue. The industry largely makes use of wild, undomesticated broodstock and thus a consortium of top producers was formed to develop a genetic improvement program to increase efficiency and sustainability of the industry. As such, the current study aimed to evaluate growth trait performance (wet weight and shell length) of four hatchery‐derived cohorts of South African abalone, across four differing production environments. No significant cohort‐by‐location effects were found for weight and length traits; however, differences were detected between respective cohorts and locations. It is concluded that a single synthetic population comprised of all hatchery cohorts under a single breeding goal is suitable for production at all test locations. Furthermore, advances in artificial reproductive technologies, genetic markers, and individual identification hold promise for genetic improvement of the species.     

偏分离条件下牙鲆生长性状QTL的主成分定位

本研究利用有丝分裂雌核发育建立牙鲆(Paralichthys olivaceus)双单倍体(Doubled Haploids,DH)群体,对牙鲆体重、全长、背鳍长、腹鳍长、体高、尾柄高、头高和躯干长共8组表型性状标准化处理后,进行主成分分析,得到可解释全部性状90.4%的表型主成分性状。然后,基于JoinMap 4、MapDisto、JoinMap 4-DistortedMap、MapDisto-DistortedMap构建4个连锁图谱,用偏分离标记矫正数量性状位点(Quantitative Trait Locus,QTL)基因型条件概率,采用Bayesian模型选择方法定位牙鲆表型主成分性状的加性QTL和上位性QTL。结果表明,用不同方法构建的遗传图谱和表型主成分性状QTL定位结果均有所不同。在图谱构建中,与基于JoinMap 4构建的图谱相比,基于MapDisto构建的图谱中偏分离标记的相对位置和遗传距离都发生了变化,甚至有5个偏分离标记没有被定位到相应的连锁群上;相对于基于JoinMap 4和MapDisto构建的图谱,经DistortedMap校正后的图谱中偏分离标记的相对位置没有发生变化,但是偏分离标记间遗传距离发生了变化。另外,在4个图谱中都检测到3个加性QTL,分别位于6号连锁群上,可解释表型变异的12.95%、14.85%、11.56%和11.76%,9号、22号连锁群上,具有负向加性效应;9号连锁群上,可解释表型变异的13.86%、13.27%、11.17%和11.25%,具有负向加性效应;22号连锁群上,可解释表型变异的5.68%、4.36%、4.97%和3.58%,具有正向加性效应。同时,分别检测到28对、19对、29对和20对上位性QTL,主要分布在6号、7号、9号、17号、20号和22号连锁群上,可解释表型主成分性状变异的2.19%~17.62%、2.40%~22.26%、2.08%~26.0%、3.16%~22.05%。研究结果表明,基于MapDisto软件构建、经DistortedMap软件包矫正后的图谱,定位结果更加准确,但仍需进一步验证。     

草鱼7个插入/缺失型突变多态性及与幼鱼生长性状关联分析

对草鱼生长性状进行数量性状基因座(quantitative trait locus,QTL)定位过程中,在15号连锁群中定位到一个与体质量相关的QTL,本研究根据已有的草鱼遗传连锁图谱和基因组序列,拟用短片段重复序列(short tandem repeat,STR)分型技术对该连锁群的3个scaffolds中插入/缺失型突变位点进行筛选,以降低分型成本,同时将筛选出的7个多态性位点与草鱼幼鱼生长性状进行关联分析。结果显示,(1)草鱼3个scaffolds中44个插入/缺失型突变位点中有17个简单重复序列(又称微卫星,simple sequence repeats,SSRs),2个位点引物设计不成功,设计的25对引物中仅22对扩增和分型成功;(2) 22个位点中仅有7对引物在4个亲本中存在多态性,直接测序结果发现,STR分型技术不仅可准确对插入/缺失型突变位点进行分型,同时可降低分型成本;(3)将7个插入/缺失型突变位点与323尾选育F2草鱼的生长性状进行关联分析发现,除了位点ID-10H和ID-41F以外,其余5个位点都与草鱼幼鱼的一个或多个生长性状显著相关,其中ID-6H与幼鱼肥满度性状显著相关,位点ID-11F、ID-15F、ID-32F和ID-39F分别与草鱼幼鱼体质量、体长、体宽和体高性状显著相关,可将以上5个与草鱼幼鱼生长性状相关的突变位点用于草鱼生长性状QTL加密和分子标记辅助育种。     

Growth and feeding in juvenile triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814), at two temperatures

Growth and feeding of juvenile triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were investigated at two temperatures of 17 and 21 °C over a 50‐day period. There were no differences in growth between triploid and diploid abalone as measured by shell length and body weight. Both triploid and diploid abalone increased in length but not in weight at 21 °C. Condition indices were similar for triploid abalone maintained at both temperatures; however, those for diploid abalone were significantly higher at 17 °C than at 21 °C. Food intake was significantly greater yet feed conversion efficiency was significantly lower in triploid than in diploid abalone. Both the feeding variables were independent of temperature. On average, diploid abalone were able to convert 1 g of dry food ingested to 0.58 g of body weight, but triploid abalone only 0.44 g.     

Feces Production and Ammonia Excretion of Pacific Abalone,Haliotis discus hannai,Fed Kelp,Laminaria japonica,in Relation to Water Temperature and Shell Length

Nitrogen excreted by aquatic animals mainly takes the form of metabolic wastes such as feces and ammonia, which is accumulated in the intensive aquaculture system and causes serious environmental contamination. So it is very important to determine the waste excretion characteristics of aquatic animals for the development of practical and nonpolluting land‐based aquaculture. Abalone has a unique feeding habit and feeding regime, different from those of finfish; abalone gnaw feed seaweed to produce feces and ammonia continuously. In this study, the rates of feces production and ammonia excretion of pacific abalone, Haliotis discus hannai, of three shell lengths (3, 5, and 7 cm) were investigated under three different temperature conditions (12, 16, and 20 C). All experiments were performed in triplicate in a semirecirculating aquaculture system. Feces were collected for 5 d, and ammonia concentrations (total ammonia nitrogen [TAN]) in the tank inlet and outlet were monitored every 4‐h interval for 24 h at the fourth day of the feces collection. The regressions for the weight‐specific feces production rate (g feces/kg abalone/d) and the weight‐specific TAN excretion rate (mg TAN/kg abalone/d) in relation to water temperature (T, C) and shell length (L, cm) were weight‐specific feces production rate = exp(1.575 ? 281.2/T² – 0.142L), r² = 0.9550, and weight‐specific TAN excretion rate = exp(5.052 ? 277.1/T² ? 0.136L), r² = 0.9598. Pacific abalone produced 108.3–111.7 g feces and excreted 3.83 g TAN/kg seaweed ingested (dry weight).     

Shell marking by artificial feeding of the tropical abalone Haliotis asinina Linne juveniles for sea ranching and stock enhancement

A method of marking abalone (Haliotis asinina Linne) for sea ranching and stock enhancement purposes was developed. Three‐month‐old abalone juveniles (11.8‐mm shell length, 0.28 g) were fed artificial diets for 1, 2, or 3 weeks. The width of the bluish‐green shell band produced by abalone juveniles was 1.7, 2.6, and 4.2 mm after 1, 2, or 3 weeks of feeding respectively. The growth and survival of juveniles fed artificial diets did not differ from that of juveniles fed the seaweed Gracilariopsis bailinae (control). Feeding the diet‐fed juveniles with the seaweed thereafter produced the natural brownish shell, thus forming a sandwiched bluish‐green band. An experimental release in outdoor tanks with natural growth of seaweeds and diatoms, and in a marine reserve showed that the shell band remained clear and distinct, indicating the usefulness of this shell marking method in sea ranching and stock enhancement of abalone.     

Growth performance of disk abalone Haliotis discus hannai in pilot- and commercial-scale recirculating aquaculture systems

The study investigated the growth performance of abalone from juvenile to marketable size in a commercial-scale recirculating aquaculture system. The rearing system consisted of 12 raceways (4.0 × 0.8 × 0.6 m) with a protein skimmer and a submerged biofilter for juveniles and 10 raceways (6.6 × 1.3 × 0.6 m) with a protein skimmer and a trickling biofilter for on-growing. Sea mustard (Undaria pinnatifida) and kelp (Laminaria japonica) were fed to the abalone. The total weight of abalone in the recirculating aquaculture system at the juvenile stage increased from 22.0 kg (average shell length 24.5 mm) to 75.5 kg (average shell length 42.5 mm) after 180 days. Feed conversion ratios increased slightly from 13.7 for the first 90 days to 16.3 thereafter. The shell growth rate of juvenile abalone between 24.5 mm and 34.8 mm was 3.4 mm month⁻¹, while for juveniles between 34.8 mm and 42.5 mm it was 2.6 mm month⁻¹. The total weight of abalone in the recirculating aquaculture system for the on-growing stage increased from 100.0 kg (average shell length 44.0 mm) to 433.3 kg (average shell length 72.7 mm) after 570 days. The feed conversion ratios for the first 173 days, the next 320 days, and the last 570 days were 19.6, 22.1, and 24.8, respectively. The growth rate of the average shell length during the on-growing period was 1.5 mm month⁻¹. Total ammonia nitrogen (TAN) concentrations were stabilized below 0.12 mg l⁻¹ in the juvenile recirculating system and 0.14 mg l⁻¹ in the on-growing recirculating system after conditioning of the biofilters.