Anguish about Irish school bullying suicide hits community
Shockwaves from the death of the Irish teenager who killed herself earlier this month are still rippling through South Hadley, the Massachusetts town where she lived, as the community struggles to come to terms with her death and with what caused it.
“It's tremendously emotionally draining on the family and the whole community right now,” said South Hadley Police Chief David LaBrie in the British Daily Mail. “It's such a sad thing.” Prince was just 15 years old when she committed suicide on Thursday January 14th. Her younger sister, who found her, was 12.
LaBrie is assisting the Northwest District Attorney's office’s investigation into Prince's death. “We are looking at all factors,” he added.
Shocking revelations about the bullying Prince endured have recently emerged. Shortly before she took her life someone sent her a text message saying: “Go kill yourself.”
According to Kevin Cullen writing in Boston.com, the bullies would call Prince a “slut,” or an “Irish slut.” The day she died, they drove past her in a car as she walked from school, shouting at her and throwing an energy drink out the window in her direction.
The bullying occurred within school as well as outside it. A friend has said Prince ran out of a math class because a girl was tormenting her, apparently jealous that a boy she liked had asked Prince to a school dance. “She was calling her names and threatening her,” the friend told the Mail. “Phoebe was probably scared. I know I would be.”
But so far, the bullies have remained unpunished, and they continue to walk the corridors of South Hadley school. Some commentators have criticized the school’s response to the situation. Barbara Coloroso, a best-selling author and consultant on bullying, was invited to speak at the school following the tragedy. “The most important thing is that kids see there are consequences that are swift and relevant, and not merely a warning,” she said, according to the Boston Herald. The school is also concerned the Prince family could sue, the paper reported.
Last week TV crews interviewed students at the school about the bullying. Right afterwards, a bully approached a girl who had spoken up, pushing her against a locker and hitting her on the head.
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The Royal Netherlands Institute of Southeast Asian and Caribbean Studies KITLV is planning to conduct a large opinion survey about the relationships within the Dutch Kingdom after the reforms of 2010, writes the Daily Herald.
Between September 14 and October 31, local interviewers will visit various addresses on the islands with a paper questionnaire. The independent and academic opinion survey will be conducted on the least-studied islands in the Dutch Caribbean; Bonaire, St. Maarten, St. Eustatius and Saba.
The interview is anonymous, can be conducted in English, Dutch, Papiamentu/ Papiamento or Spanish, and will take about 20 minutes to complete.
The results will be published later this year in various newspapers and via radio and television broadcasts. The survey is part of the research project Confronting Caribbean Challenges research project and is led by researcher Wouter Veenendaal.
The project seeks to answer the question how political reforms and intensive migrations affect historically- grounded identities and political practices on Bonaire, Statia, St. Maarten, and Saba.
The project seeks to answer this question by integrating multiple disciplines to analyse governance and identity in small-scale polities, with a particular focus on non-sovereignty, migration, and (sustainable) development.
After the dismantling of the Netherlands Antilles, the six islands remain locked in the post colonial imbroglio resulting from their rejection of full sovereignty, it is stated on the KITLV website.
“Against the advantages inherent in the asymmetrical link to the metropolis, there are constraints to the insular populations’ autonomy and identities. The constitutional reforms may have deepened the ambivalence of Antillean citizens and politicians towards the Netherlands, while high levels of immigration raise other questions about insular belonging. The Confronting Caribbean Challenges project will analyse the intersection of (recent) political reforms, often intensive migrations, and current practices of governance on four of these islands,” it is stated on the website.
The focus is on the least studied islands of the former Netherlands Antilles. The project utilizes a three tiered multi-disciplinary framework linking the past (historical development of hybrid identities), the present (governance, migration and citizenship issues), and the future (sustainable tourism capitalizing on natural and historical heritage).
The three sub-projects are to combine an historical perspective, contemporary empirical data and fresh insights into the literature on governance and identity in small-scale polities, emphasizing the variables of non-sovereignty and high intensity migration, it was explained.

domain: geography


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- Open Access
Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation
Microbial Cell Factories volume 14, Article number: 70 (2015)
Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation.
Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw−1 h−1, and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw−1 h−1, much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene.
This study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.
Bio-ethanol, currently blended in gasoline, is primarily produced from microbial fermentation of food-based sugars, such as corn starch and cane sugar. Bioethanol derived from food-based substrates is unsustainable as it competes with our food chain and may cause human starvation. On the other hand, ethanol production from the abundant lignocellulosic biomass, such as agricultural wastes and forest waste, promises a sustainable green fuel. However, effective conversion of xylose, the second abundant sugar after glucose in lignocellulosic biomass, is necessary to make this process more economically feasible and efficient . Saccharomyces cerevisiae is an attractive host to produce biofuels and industrial chemicals from lignocellulosic biomass due to its robustness and fast fermentation rate . However, it cannot naturally utilize xylose . Xylose isomerase (XI) naturally catalyzes the bacterial conversion of xylose to xylulose, which is further metabolized to ethanol through central metabolic pathways. One of the essential metabolic engineering modifications for effective xylose fermentation by S. cerevisiae is expressing an XI with high activity. However, the species boundary makes most of eubacterial xylose isomerases fail to be functionally expressed in yeast.
Besides XI pathway, xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway from Pichia stipitis (Scheffersomyces stipitis) was also engineered in S. cerevisiae for the construction of xylose-fermenting yeast [4–7]. In XI pathway, xylose is firstly isomerized into xylulose through one-step catalysis by XI and xylulose was then phosphorylated into xylulose 5-phosphate by xylulokinase. The latter is the intermediate metabolite of pentose phosphate pathway and can be further converted into ethanol through glycolysis . In XR and XDH pathway, xylose is firstly reduced into xylitol by XR, and xylitol is then oxidized into xylulose by XDH . This pathway has been successfully constructed in S. cerevisiae; however, xylitol accumulation is a problem. This is caused by the uncoupled cofactor consumption of NADPH-preferred XR and NAD+-preferred XDH . Although anaerobic xylose-fermenting yeast was successfully constructed by the overexpression of NADH-preferred XR or NADP+-preferred XDH generated through protein engineering [1, 8], xylitol accumulation was still a problem and the specific xylose consumption was not as efficient as XI expressed strains [9–11]. Therefore, xylose isomerase pathway is still the method of choice for the construction of efficient xylose-fermenting yeast.
XI commonly exists in bacteria. However, because of unknown molecular barrier between yeast and bacteria, most Proteobacteria XI could not be expressed in S. cerevisiae functionally. It was found that XIs from Escherichia coli, Bacillus subtilis, and Streptomyces rubiginosus were expressed as the insoluble form in S. cerevisiae [12–14]. XI from Thermus thermophilus was firstly reported to be actively expressed in S. cerevisiae. However, its optimal reaction temperature was 85 °C, and at 30 °C it only demonstrated about 4 % of the maximum activity, which was insufficient for fast xylose consumption . The first reported XI that guaranteed effective xylose fermentation in S. cerevisiae at 30 °C was from the anaerobic fungus Piromyces sp. E2, and the recombinants generated from XI gene transformation and adaptive evolution consumed xylose at the specific rate of 0.18 to 1.87 g−1 xylose g−1 biomass h−1 [6, 7, 15]. XIs from another anaerobic fungus Orpinomyces sp. ukk1 , several Bacteroid bacteria [17, 18], Clostridium phytofermentans , Ruminococcus flavefaciens  and Prevotella ruminicola  have also been successfully used to construct xylose-fermenting yeast. Information on the active XIs in yeast is scattered, and it would be instructive to rational selection of XIs to efficiently construct xylose-fermenting yeast if the evolutionary relationship among the active XIs can be elucidated.
In this study, phylogenetic analysis of the active XIs was conducted and it was found that majority of the reported active XIs were clustered in mammal gut Bacteroidetes group. This suggests that other XIs clustered in this group might also be able to function in S. cerevisiae. In order to confirm this, four different deposited XI genes from mammal gut Bacteroidetes group were tested for their functional expression in yeast. In addition, two XIs were cloned from snail manure metagenomic DNA by degenerate PCR and thermal asymmetric interlaced (TAIL) PCR, and they were used as the reference. The two cloned XIs were clustered with XIs from environmental Bacteroidetes bacteria. As expected, only XIs from the mammal gut Bacteroidetes group were expressed in S. cerevisiae with activity. Through combined optimization of xylose metabolic pathways and adaptive evolution in xylose medium, recombinant yeast strains containing mammal gut Bacteroidetes XIs were generated and all of them showed efficient xylose fermentation.
Results and discussion
Sequence analysis and cloning of xylose isomerase (XI) from environmental mega-genome
With the fast development of genome-sequencing technique, increasing numbers of XI sequences were deposited in public nucleotide/protein databases, such as Genbank, ENA and Uniprot. For the exploration of XIs that are potentially functional in yeast, XI sequences were retrieved and their phylogenetic relationship was analyzed. XIs were grouped into two classes (class I and class II) . Class I XIs are distinguished from class II XIs by having a shorter N-terminal, and T. thermophilus XI, the first XI actively expressed in S. cerevisiae, belongs to class I. However, another class I enzyme from S. rubiginosus was misfolded when expressed in yeast. Class II represents a large number of XI families, and the relationship of XIs in phylogenetic tree is highly consistent with the taxonomic orders (Fig. 1). However, only a few of them are active in yeast. Currently, most of the active XIs in yeast belong to Class II. The phylogenetic tree shows that XI expressed actively at 30 °C in yeast are in three branches, Bacteroidetes, C. phytofermentans-R. flaverfaciens and higher eukaryotes (Ciona and Arabidopsis) (Fig. 1), and majority of the active XIs are from gut bacteria (or fungi) of mammals. This suggests that the active expression of microbial xylose isomerase in S. cerevisiae might be related to the diverse evolutionary events of mammalian gut commensals where xylan-degrading microorganisms are abundant .
Among the active XIs in yeast, XIs originated from the phylum Bacteroidetes represent a significant number. XIs from gut Bacteroiedetes of mammals are categorized into a group different from environmental Bacteroidetes XIs, and XIs from fungi, Piromyces and Orpinomyces, are also clustered in this group. Therefore, it is very likely that other XIs clustered in this group might also be able to function in S. cerevisiae. Based on this hypothesis, in this study, the deposited gene sequence encoding XIs from Bacteroides vulgatus, Alistipes sp. HGB5, Paraprevotella xylaniphila, and Tannerella sp. 6_1_58FAA_CT1 from the mammal gut Bacteroidetes group were selected and tested for the construction of xylose-fermenting yeast.
In order to isolate novel functional XI genes in yeast, XI sequences from metagenomic DNA of snail manure and forest soil were directly amplified through degenerated PCR and TAIL-PCR. Interestingly, degenerated PCR products of snail manure sample and forest soil sample shared the same sequences, and the deducted amino acid sequences were related to environmental Bacteroidetes group (Fig. 1). Eleven unique sequences in total were amplified, but all were clustered in environmental Bacteroidetes group (data not shown). The upstream and downstream sequences of two degenerated products (SmmXylA1 and SmmXylA10) were amplified successfully through TAIL-PCR. Based on the sequencing results, the full-length genes of SmmXylA1 and SmmXylA10 (SEQ. A1 and SEQ. A2) were amplified directly from metagenomic DNA of snail manure sample and were also tested for their functional expression in yeast.
Expression of the xylose isomerase from two clades of Bacteroidetes xylose isomerases
B. vulgatus is a xylan-degrading bacterium habituating in human gastrointestinal tract, and its genome sequence has been released . B. vulgatus XI shares 81 % identical amino acid sequence and 38 % identical nucleic sequence with Piromyces sp. E2 XI. The full length gene was directly synthesized according to the deposited sequence under EMBL-ENA accession No. ABR41556.1 without codon usage optimization so that the potential of the original XI in its functional expression in S. cerevisiae can be evaluated. B. vulgatus XI gene was fused with TDH3 promoter and PGK1 terminator. In addition, B. vulgatus XI gene, Piromyces sp. E2 XI (EMBL:AJ249909.1) gene and the two XI genes cloned from metagenomic DNA were cloned between TEF1 promoter and CYC1 terminator. Notably, only B. vulgatus and Piromyces sp. E2 XIs were expressed with activities, and the strains harboring the metagenomic XI genes and the reference strain carrying the empty vector almost showed no activity (Fig. 2).
Active expression of B. vulgatus XI in S. cerevisiae is expected because its amino acid sequence is close to those of XIs from Piromyces sp. E2, Bacteroides stercoris  and P. ruminicola , which are all clustered with the XIs from mammal gut Bacteroidetes group. Interestingly, although the original B. vulgatus XI gene sequence had a low value (0.146) of codon adaptation index (CAI) to S. cerevisiae, the demonstrated activity of B. vulgatus XI was significantly high (Fig. 2), revealing the potential function of this XI in S. cerevisiae. While B. vulgatus XI under the promoter of TDH3 (JUK51a_1) displayed much higher activity than Piromyces sp. E2 XI under TEF1 promoter (JUK52a), under TEF1 promoter B. vulgatus XI (JUK51a_2) and Piromyces sp. E2 XI (JUK52a) exhibited comparable activities (Fig. 2). The fact that TDH3 promoter is a stronger promoter than TEF1 promoter  might contribute to the high XI activity in JUK51a_1. For the development of background strain, strain JUK51a_1 harboring B. vulgatus XI under TDH3 promoter (Table 1) was used. Consistent with the phylogenetic analysis (Fig. 1), the isolated metagenomics XI genes, SmmXylA1 and SmmXyl10, belonging to group of environmental Bacteroidetes, were not active in S. cerevisiae (JUK53a and JUK54a in Fig. 2).
Improving xylose-fermenting background yeast strain using recombinant yeast strain harbouring B. vulgatus XI
Overexpressing of XI with high activities alone could not render the effective fermentation of xylose . Therefore, xylose metabolic pathway in the above background S. cerevisiae strain was optimized by overexpressing xylulokinase (XKS1) and the four enzymes from non-oxidative pentose phosphate pathway, transaldolase (TAL1), transketolase (TKL1), ribose-5-phosphate isomerase (RKI1), and ribulose 5-phosphate 3-epimerase (RPE1), and disrupting the aldose reductase gene gre3. Host strain (JUK36a) was generated, and the recombinant yeast strains harboring B. vulgatus XI strains JUK51a_1 (XI controlled by TDH3 promoter) and JUK51a_2 (XI controlled by TEF1 promoter) exhibited aerobic growth on xylose. On the other hand, the control strain without XI genes (JUK50a) could not grow on xylose (data not shown). Adaptive laboratory evolution in xylose medium under the anaerobic conditions or using a respiration-deficient strain was shown effective to reinforce xylose catabolism to ethanol production [7, 10, 15, 21]. Therefore, before more XIs from the mammal gut Bacteroidetes cluster were tested, a respiration-deficient strain (JUK39a) was generated from JUK36a by disrupting cyc3, which encodes holocytochrome c synthase and attached heme to apo-cytochrome c , and it could not grow on glycerol (data not shown). Plasmid pJFX11-TDH3p was transformed to JUK39a to generate the respiration-deficient strain JUK61a. Strain JUK61a exhibited the delayed growth on xylose and the growth rate increased by adaptive evolution through continuous transferring cultivation in synthetic minimal medium (SMM) containing 6.7 g l−1 yeast nitrogen base (YNB) and 20 g l−1 xylose at pH 6.0 (Fig. 3a).
Unlike strain JUK51a_1, for which xylose metabolism is dependent on respiration, strain JUKx11a, isolated from the continuous transferring cultivation of strain JUK61a on xylose, exhibited efficient xylose fermentation (Fig. 4). In batch aerobic cultivation, ~15 g l−1 xylose was consumed in 24 h, and ~6 g l−1 ethanol and ~1.4 g l−1 glycerol were produced. Ethanol yield reached 0.396 g g−1 consumed xylose. This strain was maintained in SMM with 20 g l−1 xylose and it was stable for xylose fermentation after more than 20 times of transferring. Similar results were obtained for anaerobic batch cultivation using strain JUKx11a indicating oxygen supply to this strain is not essential to this respiration-deficient strain (data not shown).
Saccharomyces cerevisiae strain JUK51a_1 (Table 1) was also anaerobically evolved in a chemostat by continuous diluting cultivation until its dilution rate was increased from 0.01 to 0.11 h−1 (Fig. 3b). A fast growing strain was isolated and the plasmid of pJFX11-TDH3p (URA3) was lost by streaking on 5-fluoroorotic acid (5-FOA) plates  to obtain strain JUK36a1 (Table 1). Plasmid pJFX11-TDH3p was also lost from the evolved respiration-deficient strain JUKx11a similarly to obtain strain JUK39a1 (Table 1). Both strains JUK36a1 and JUK39a1 were used as hosts for further XI testing.
Parallel xylose utilization and cell growth testing of yeast strains expressing XI genes from mammal gut Bacteroidetes cluster
Based the background strains JUK36a1 and JUK39a1, more XIs from the mammal gut Bacteroidetes cluster (Fig. 1) were tested and compared. Beside B. vulgatus XI gene, XI genes from Tannerella sp. 6_1_58FAA_CT1, P. xylaniphila, Alistipes sp. HGB5 and Orpinomyces sp. ukk1 (used as a positive control)  were individually introduced into the evolved background strains under the control of PGK1 promoter. The five XIs respectively have an identity of 95 % (Orpinomyces sp.), 82 % (P. xylaniphila), 81 % (Tannerella sp.), 81 % (B. vulgatus) and 76 % (Alistipes sp.) to the amino acid sequence of Piromyces sp. XI.
SMM supplied with 40 g l−1 xylose was used in batch cultivation of 36a and 39a strains. Aerobic cultivation was performed in 24-well plates with 600 μl media at 218 rpm and 30 °C. Duplicate wells were used for each strain. Biomass was monitored by measuring the absorbance at the wavelength of 595 nm.
The concentrations of glucose, xylose, xylitol, glycerol, acetate, and ethanol were determined using HPLC and results are listed in Fig. 5. Notably, all 36a and 39a strains expressing the above five different xylose isomerase genes grew well in the SMM containing xylose. Interestingly, all 36a strains grew almost equally fast, indicated by very similar maximal specific growth rate of about 0.2 h−1 (Fig. 5), with strain 36a(HGB5) containing Alistipes sp. HGB5 XI gene presenting the highest specific growth rate of 0.2015 h−1, and strain 36a(Bvu) expressing B. vulgatus XI gene showed very similar specific growth rate of 0.2009 h−1, both were slightly higher than that (0.1977 h−1) obtained by the control strain 36a(XIq) harboring Orpinomyces sp. ukk1 XI gene. Strain 36a(TAA) carrying the Tannerella sp. 6_1_58FAA_CT1 XI gene and 36A(YIT) containing the P. xylaniphila XI gene presented equal specific rate of 0.1850 h−1, slightly lower than other three strains. The specific rate values were much comparable to that obtained for S. cerevisiae expressing codon-optimized P. ruminicola XI gene . In the latter case, a specific rate of 0.23 h−1 was obtained after adaptive evolution of the recombinant strain in SMM containing xylose. Apparently, the relatively high specific rates obtained for the recombinant 36a strains is attributed to the background strain, JUK36a1, which has been anaerobically evolved in xylose medium. These results demonstrated that host strain anaerobic adaptive evolution in xylose medium followed by XI gene transformation might be an effective way for the screening of novel functional xylose isomerase genes in yeast. Orpinomyces sp. ukk1 is a mammal gut fungus and its XI gene is very well known for its functional expression in S. cerevisiae . The comparable performance of xylose utilization between the recombinant strains harboring the four selected XI genes from the mammal gut Bacteroidetes cluster to the control strain points out the potential of such bacteria XI genes in functional expression in S. cerevisiae and xylose-fermenting yeast construction.
On the other hand, cell growth rates varied among the 39a strains. Obviously, the knock-out of cyc3 gene, which encodes holocytochrome c synthase and attached heme to apo-cytochrome c, was able to differentiate the active expression of different xylose isomerase genes. Strain 39a(TAA) harboring the Tannerella sp. 6_1_58FAA_CT1 XI gene demonstrated the fastest growth with 0.1584 h−1 specific growth rate, followed by 39a(YIT) carrying the P. xylaniphila XI gene with about 0.1145 h−1 specific growth rate and strain 39a(Bvu) containing the B. vulgatus XI gene with specific growth rate of 0.1104 h−1. Notably, the control strain 39a(XIq) expressing the Orpinomyces sp. ukk1 XI gene in this case displayed the lowest specific growth rate of 0.0882 h−1 and strain 39a(HGB5) containing Alistipes sp. HGB5 XI gene displayed slightly higher specific growth rate of 0.1006 h−1. The above results suggest that Tannerella sp. XI is more favorable to cell growth on xylose using the respiration-deficient strain JUK39a1 as the host. In general, 36a strains grew faster than 39a strains. The specific growth rates of 36a strains were about 0.20 h−1 and those for 39a strains were about 0.10 h−1, except strain 39a(TAA) presenting a specific growth rate of about 0.16 h−1. The final OD595 values of 36a strains were above 1.6, whereas those for 39a strains were below 1.4.
Xylose fermentation by recombinant 36a and 39a strains
Better aerobic cell growth does not always correlate to better xylose fermentation. In order to identify the best xylose-fermenting recombinant yeast strains, in this part of the work, batch fermentation of recombinant 36a and 39a strains was performed in a 125 ml flask plugged with a rubber stopper with a syringe tip outlet at 30 °C and 200 rpm. SMM containing 40 g l−1 xylose was used in the cultivation. Interestingly, all 36a strains were able to utilize xylose as the sole carbon source and produced significant amount of ethanol (Fig. 6). This is different from the case of recombinant strains JUK51a_1 and JUK51a_2, whereby although both strains were able to grow in SMM containing xylose, no xylose-fermentation was observed. The ability of xylose-fermentation of the 36a strains was arisen from the anaerobic adaptive evolution of strain JUK51a_1 in xylose medium. Likewise, 39a strains were also able to grow in SMM containing xylose and produce ethanol, which otherwise was attributed to the anaerobic adaptive evolution of JUKx11a. For all the 36a strains tested, notably, strain 36a(HGB5) harboring Alistipes sp. HGB5 xylose isomerase gene was able to utilize xylose more rapidly than other strains and produced more ethanol (Fig. 6). On the other hand, for all 39a strains, strains harboring XI genes from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1 and P. xylaniphila almost displayed almost identical rates of xylose consumption and ethanol production, with the control strain having the Orpinomyces sp. ukk1 XI gene showed the slowest xylose utilization and ethanol production rates (Fig. 6). Apparently, for strain 36a(HGB5) and 39a(TAA), the high rates of xylose consumption and ethanol production correlated well with the better cell growth and higher cell density. Correspondingly, the low xylose consumption and ethanol production by 39a(XIq) was due to its slow cell growth and lower cell density. However, the production of glycerol did not correlate with its cell growth. Strain 36a(HGB5) exhibited the lowest glycerol production among all the 36a strains, whereas strain 39a(TAA) displayed moderately high glycerol production among all the 39a strains.
Fermentation parameters of 36a and 39a strains at 56 h are listed in Table 2 for detailed comparison, and the average specific xylose consumption, ethanol and glycerol production rates were calculated according to Peng and his colleagues . Among all the 36a strains, notably, 36a(HGB5) presented the highest ethanol titer and xylose conversion, indicating its fastest xylose utilization rate. This might be associated with its highest cell growth rate revealed by its high final biomass concentration. Correspondingly, its specific ethanol productivity was lower than the rest of strains, except the control strain 36a(XIq). Interestingly, the control strain also displayed the lowest ethanol yield of 0.365 g g−1, corresponding to about 72 % of the theoretical value. This arose primarily from its lower ethanol titer than the rest strains (Table 2). The main by-product for 36a strain fermentation was glycerol, with its specific production rate of about 0.02 to 0.13 g gcdw −1 h−1. Very minimal amount of xylitol and acetic acid were produced with their specific production rates being lower than 0.01 g gcdw −1 h−1 (data not shown). For the 39a strains, the highest ethanol titer was obtained by 39a(Bvu), whereas the highest xylose conversion was obtained by strain 39a(TAA) with the 2nd highest ethanol titer. This is largely due to its high cell growth rate (Fig. 6) and cell density. Further analysis of the specific rates of xylose consumption and ethanol production suggest that strain 39a(HGB5) is the most potential by showing the highest rates, just below the control strain 39a(XIq). Strain 39a(Bvu) showed slightly lower specific rates of xylose consumption and ethanol production. The fact that 39a strains containing B. vulgatus and Alistipes sp. HGB5 XI genes exhibited moderately high cell growth rate and higher specific rates of xylose consumption and ethanol production recommends that these two XI genes are the most potential in xylose-fermenting yeast construction using JUK39a1 as the host. Similar to 36a strains, the main by-product for 39a strains was glycerol with specific rate of production of about 0.03 g gcdw −1 h−1. Production of xylitol and acetic acid was very minimal with both specific production rates being smaller than 0.01 g gcdw −1 h−1 (data not shown).
Strain 39a(TAA) demonstrated the lowest specific ethanol productivity (0.213 g gcdw −1 h−1) with the highest being 0.295 g gcdw −1 h−1 obtained by the control strain 39a(XIq). Interestingly, such specific ethanol productivity values for 39a strains were much higher than those reported by Peng et al.  and Hector et al. , where they were below 0.100 g gcdw −1 h−1, and were comparable to those obtained by 36a strains ranged from 0.104 to 0.134. Our recombinant strains were constructed by strain adaptive evolution in xylose medium followed by XI gene transformation; whereas the recombinant strains by Peng et al  and Hector et al.  were constructed by XI gene transformation followed by adaptive evolution in xylose medium. Much comparable cell growth (Fig. 5) and fermentation performance (Table 2) were obtained, suggesting that the approach adopted in this work is effective in rapid screening of functional XI genes and efficient in construction of xylose-fermenting yeasts.
It is worthwhile noting that consistent ethanol yield of about 0.4 g g−1 was obtained for all the 39a strains tested, with the highest being 0.419 g g−1 for strain 39a(Bvu) and the lowest being 0.401 g g−1 for strain 39a(TAA), corresponding to 82.2 % and 78.6 %, respectively, of the theoretical yield. The control strain 39a(XIq) in this case obtained the highest ethanol yield of 0.409 g g−1, corresponding to 80.2 % of the theoretical yield. The significant difference of ethanol yield for strains 36a(XIq) and 39a(XIq) confirms that respiration-deficient background strain is more favorable for the expression of fungus Orpinomyces sp. XI gene. Moreover, all the 39a strains consumed xylose faster and produced more ethanol than 36a strains, further proving that respiration-deficient strain is more favorable for xylose fermentation although anaerobic adaptive evolution is able to enhance it for respiration-efficient 36a strains. The favorable xylose utilization and ethanol production by 39a strains is ascribed to the anaerobic nature of these mammal gut bacteria.
Xylose isomerase genes from two phylogenetic groups (mammal gut Bacteroidetes and environmental Bacteroidetes) were tested in S. cerevisiae strain with downstream xylose metabolic pathway optimization. Xylose isomerase from mammal gut microorganisms, B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Alistipes sp. HGB5 and P. xylaniphila, were actively expressed, and the two Bacteroidetes-related genes isolated from environmental metagenome did not exhibit activities. Expression of the above XI genes from the above listed mammal gut bacteria in a respiration-efficient host, S. cerevisiae JUK36a1, resulted in similar growth rate under aerobic conditions. On the other hand, the respiration-deficient host, JUK39a1, resulted in varied growth rates for strains harboring XI genes from different mammal gut bacteria. The expression of Tannerella sp. 6_1_58FAA_CT1 XI gene resulted in the fastest cell growth rate, maximal xylose conversion, and highest ethanol production under anaerobic cultivation conditions. Respiration-deficient S. cerevisiae strain carrying B. vulgatus and Alistipes sp. HGB5 XI genes presented moderate cell growth and relatively high specific ethanol productivity of 0.277 and 0.283 g gcdw −1 h−1, respectively. They are most potential in xylose-fermenting yeast construction. All respiration-deficient strains (39a strains) displayed higher rates of xylose consumption and ethanol production than 36a strains, confirming that cyc3 gene knock-out is favorable. In addition, this work also demonstrated that host strain adaptive evolution in xylose medium followed by XI gene transformation is effective in rapid functional XI gene screening in S. cerevisiae and is efficient in xylose-fermenting yeast construction.
Cloning of xylose isomerase genes from environmental metagenomic DNA
Primers used in this work are listed in Additional file 1. Metagenomic DNA from snail manure and soil from Singapore natural forest was extracted using FastDNA® Spin Kit for Soil (MP Biomedicals) and purified using E.Z.N.A. ® Cycle-Pure Kit (Omega Bio-tek). Degenerate primers were designed based on xylose isomerase conservative region (Additional file 1). Primers MetXIconss and MetXIconsa were used to amplify partial sequences of xylose isomerase from snail manure metagenomic DNA (Smm) and soil metagenomic DNA (Som). The PCR product of partial sequences of xylose isomerase was cloned into T-vector and sequenced. The 5′ and 3′ flanking regions of xylose isomerase from metagenomic DNA were obtained through thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) using LA Taq DNA polymerase (TAKARA), and arbitrary degenerated primers (AD1, AD2, AD3 or AD4) and nested gene-specific primers. TAIL-PCR was performed according to the previously reported protocol  and the TAIL-PCR products were sequenced to generate the whole gene sequences. Primers designed based on 5′ and 3′ regions of xylose isomerase were used to amplify the full-length genes directly from metagenomic DNA.
Strains and plasmids used in this work are listed in Table 1.
The gene fragments, FBA1p-TKL1(1318) (recombinant arm targeting to TKL1 open reading frame, ORF), TKL1(-501,-286) (recombinant arm targeting to TKL1 promoter), PDC1p-TAL1-TAL1t, ADH1p-RKI1-RKI1t, TPI1p-RPE1-RPE1t and PGK1p-XKS1-XKS1p were amplified using genomic DNA of S. cerevisiae ATCC 24860 and fused through overlapping extension polymerase chain reactions (PCR). G418 resistant marker loxP-KanMX4-loxP was amplified from plasmid pUG6. These DNA fragments were sequentially cloned into pUC19 to generate the plasmid pJPPP-XK (Fig. 7).
The coding sequence of B. vulgates xylose isomerase was synthesized according to the sequence under EMBL-ENA access No. ABR41556.1 and fused with TDH3 promoter and PGK1 terminator from S. cerevisiae through overlapping extension PCR to generate DNA fragment TDH3p-BvuXylA-PGK1t. Auxotrophic marker URA3 was cut out from pUG72 by XbaI and NdeI. DNA fragments of TDH3p-BvuXylA-PGK1 and URA3 were sequentially cloned into SalI/PstI sites and SpeI/NdeI sites of pRS327 to generate pJFX11-TDH3p (Fig. 7). The yeast expression vector pJFE11 was constructed by replacing GAL1 promoter of plasmid pYES2 by TEF1 promoter. The coding sequences of B. vulgates XI, Piromyces sp. XI, SmmXylA1 and SmmXylA10 were cloned into BamH/XhoI sites of pJFE11 (Fig. 7) between TEF1 promoter and CYC1 terminator to generate pJFX11, pJFX12, pJFX13 and pJFX14, respectively (Table 1).
S. cerevisiae haploid strain was isolated from the diploid strain S. cerevisiae ATCC 24860. The LiAc/ssDNA/PEG solution was used for yeast transformation . Disruption cassettes of ura3, gre3 and cyc3 with G418 resistant marker loxP-KanMX4-loxP were amplified from pUG6 . These disruption cassettes were transformed into yeast to generate the strains with relevant genotypes. Xylulokinase and non-oxidative pentose phosphate pathway were overexpressed by transforming SwaI-digested pJPPP-XK (Fig. 7). The KanMX4 markers for disruptions of ura3, gre3 and cyc3 and pJPPP-XK integration were separately removed by transforming a Cre plasmid pSH47 (URA3) and inducing Cre expression in YP galactose broth. Later on, pSH47 (URA3) was lost by cultivation on 5-FOA plates .
Synthetic minimal media (SMM, Yeast nitrogen base, YNB, 6.7 g l−1, pH 6.0) supplied with glucose and/or xylose was used in batch cultivation. Aerobic cultivation was performed in 24-well plate with 600 μl medium at 218 rpm and 30 °C and cell growth was monitored by measuring the absorbance at the wavelength of 595 nm. Oxygen-limited cultivation was performed in 125 ml Erlenmeyer flasks, plugged by rubber stoppers with syringe needle outlets, with 60 ml media incubated at 200 rpm and 30 °C. Cells in late exponential-phase in SMM with 20 g l−1 xylose were harvested by centrifuge and inoculated into the cultivation medium for fermentation evaluation. Cell biomass was monitored by measuring the absorbance at the wavelength of 600 nm.
Adaptive transferring cultivation was performed in a 125 ml Erlenmeyer flasks, plugged by rubber stoppers with syringe needle outlets, containing 20 ml SMM with 20 g l−1 xylose incubated at 200 rpm and 30 °C. In each batch of adaptation, the initial OD600 was set as 0.2, and when OD600 reached about 2.5, cells were inoculated into a new batch with an initial OD600 of 0.2 for further adaptation. Finally, single clones were separated by streaking on the SMM xylose plate, and the biggest clone was selected and named as JUKx11a after seven day incubation.
Continuous evolutionary cultivation was performed in a 200 ml feeding bottle with 150 ml SMM supplied with 12 g l−1 xylose as the carbon source and a stirring magnetic bar at 30 °C, under anaerobic conditions. The cultivation was initiated by inoculating the JUK51a_1 cells cultivated in the aerobic conditions. The fresh media was continuously added through a feeding pump. Biomass was monitored by measuring the absorbance at wavelength of 600 nm. According to cell growth, the dilution rate was increased gradually from 0.01 to 0.11 h−1.
The concentrations of glucose, xylose, xylitol, glycerol, acetate, and ethanol were determined using HPLC with an Aminex HPX-87H ion exchange column (Bio-Rad) at 60 °C, and a refractive index detector; 5 mmol l−1 H2SO4 was used as the mobile phase with a flow rate of 0.6 ml min−1 . All aerobic cultivation and xylose fermentation experiments were conducted in duplicate, and the average value with standard deviation was reported.
Xylose isomerase activity assay
Cells were cultivated in SMM supplemented with 20 g l−1 xylose and were harvested at OD600 of 2.5. Cell-free extracts were prepared in 100 mM tris-HCl buffer (pH 7.5) using a glass bead beater and protease inhibitor cocktail set IV (Merck) was added. The xylose isomerase activity of the cell extracts was determined at ambient temperature (25 °C) using the UV-VIS Spectrophotometer 1240 (Shimadzu). The 1-ml reaction mixture contained 100 mmol l−1 Tris–HCl buffer (pH 7.5), 10 mmol l−1 MgCl2, 500 mmol l−1 xylose, 1 U of sorbitol dehydrogenase (Roche), 0.15 mmol l−1 NADH, and 0.05 ml of the cell extract . Protein concentration was measured using the Coomassie protein assay kit (Thermo Scientific). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of coenzyme/min, and the specific activity was expressed in units per milligram of protein.
- ADH1 :
American Type Culture Collection
Codon adaptation index
- CYC1 :
- cyc3 :
Holocytochrome c synthase gene
Cell dry weight
European Molecular Biology Laboratory – European Nucleotide Archive
- FBA1 :
- gre3 :
Aldose reductase gene
- NADP+ :
Oxidized nicotinamide adenine dinucleotide phosphate
Reduced nicotinamide adenine dinucleotide phosphate
- NAD+ :
Oxidized nicotinamide adenine dinucleotide
Reduced nicotinamide adenine dinucleotide
- PDC1 :
- PGK1 :
3-phosphoglycerate kinase promoter
- RKI1 :
- RPE1 :
Ribulose 5-phosphate 3-epimerase
Synthetic minimal medium
Thermal asymmetric interlaced polymerase chain reaction
- TDH3 :
- TAL1 :
- TKL1 :
- TEF1 :
Transcription elongation factor
Yeast episomal plasmid
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The authors are grateful for the financial support to this work provided by the Science and Engineering Research Council of the Agency for Science, Technology and Research (A*STAR) and National Research Foundation, Singapore.
PCT specifications were filed on 16 December 2014. Patent Cooperation Treaty Application No. PCT/SG2014/000600.
BP, SH, and TL carried out all the experiments, acquired the data and BP drafted the manuscript. AG designed the study, performed results interpretation and analysis, and critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.
Mr. Bingyin Peng was a Research Scientist at School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore when he conducted this research. He was working on the metabolic engineering of yeast strains for cellulosic ethanol production.
Mr. Shuangcheng Huang is currently a Research Assistant at School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore. He is working on the metabolic engineering of yeast strains for cellulosic ethanol and biochemical production.
Miss Tingting Liu is currently a Research Scientist at School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore. She is working on the metabolic engineering of yeast strains for cellulosic ethanol and biochemical production.
Dr. Anli Geng is currently the Assistant Director (Technology) and Senior Lecturer at School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore. She is the Principal Investigator of the yeast metabolic engineering research.
About this article
Cite this article
Peng, B., Huang, S., Liu, T. et al. Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation. Microb Cell Fact 14, 70 (2015). https://doi.org/10.1186/s12934-015-0253-1
- Xylose isomerase
- Mammal gut
- Adaptive evolution
- Saccharomyces cerevisiae
- Xylose fermentation
- Cellulosic ethanol

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West Australia mine desalination plant review published
A public environmental review (PER) was issued on 26 September 2011 for a 35,000 m³/d seawater desalination plant at Cape Riche in Western Australia, 90 km northeast of Albany.
Dry Australian state mulls starting up desalination plant
A A$ 4 billion (US$ 2.93 billion) desalination plant in Australia's Victoria state could make its debut as parts of the state are suffering their driest period on record.
Fourth IDE Progreen unit to go to Australian resort
IDE Technologies is to install its Progreen desalination system - the fourth installation worldwide - at a five‑star island resort on Australia's Great Barrier Reef to replace an existing thermal desalination plant.
Australian government to fund stormwater reuse
The government of Australia has invited funding applications by 7 December 2011 for support of stormwater harvesting and reuse projects.
US$ 2.9 billion Melbourne desalination contract goes to Degremont group
The Suez/Degremont-led consortium Aquasure has been awarded the contract to build the 150 million m³/year seawater desalination plant for Melbourne, Australia.
Sydney Desalination Plant leased for US$ 2.3 billion
The New South Wales state government in Australia accepted on 10 May 2012 a binding offer for refinancing the Sydney Desalination Plant (SDP) from a consortium of Ontario Teachers' Pension Plan (OTPP) and Hastings Funds Management.
Australia failing to meet wastewater reuse targets
Australia is falling well behind in the targets it has set itself for wastewater reuse, according to a study carried out for the Department of Sustainability, Environment, Water, Population & Communities by Marsden Jacob Associates (MJA).
Reuse highlighted in WSAA urban water roadmap
The volume of water being recycled by the largest water utilities in Australia has increased by 117% since 1999/2000. In addition, it is estimated that, by 2013, 460 million m³/year will be supplied to Australia's growing cities by desalination plants.
Veolia given Sydney wastewater reuse licence
New South Wales premier Nathan Rees announced on 7 May 2009 that Veolia Water has been issued the first licence under the Water Industry Competition Act 2006 to construct, maintain and operate a new recycled water plant at Fairfield, Sydney Australia, as part of the Rosehill Recycling Scheme.
Olympic Dam desalination EIS released by BHP Billiton
The desalination plant in South Australia to feed BHP Billiton's Olympic Dam mining development moved a step closer when the company released the final supplementary environmental impact statement (SEIS) for the project on 13 May 2011.
Water rights row could delay Western Australia desalination plant
A row over water rights in the north-west of Australia could lead to delay in the construction of the West Pilbara Seawater Desalination Plant, despite assurances in February 2011 from Western Australia's (WA) state government that the project was on track.
More desalination and reuse potential in Western Australia
Western Australia's Water Corporation is considering up to three seawater reverse-osmosis desalination plants along the southern coast of its area, known as Lower Great Southern, as part of a potential portfolio of water resources for the next 30 years.
Australia's desalination centre sited at Murdoch University
Australia's new National Centre of Excellence in Desalination is to be hosted by Murdoch University's Rockingham campus as a result of a Aus$ 20 million (US$ 15.5 million) award from the federal government.
NCED/MEDRC workshop will look at red tide and desalination
A joint workshop on the dangers of the red-tide marine phenomenon for desalination plants is to be held in Oman by the National Centre for Excellence in Desalination Australia and the Middle East Desalination Research Center.
Xylem appoints new emerging markets head
Water technology firm, Xylem, has appointed Steven Leung as senior vice president and president, emerging markets, In this newly created position, Leung will lead the company's commercial operations in China, greater Asia, India, the Middle East, Africa and Latin America, as well as Australia and New Zealand.
Scientists to probe outlet brine risks to sealife
Scientists have been commissioned to investigate whether brine waste from two proposed 150 Ml/d seawater reverse osmosis desalination plants could damage or alter the marine environment along the KwaZulu-Natal coastline in South Africa.
Ex-Adelaide desalination chief joins Singapore's Hyflux
Milind Kumar, formerly project director of the Adelaide Desalination Plant for South Australia Water, has been appointed to a senior position in Singapore group Hyflux Ltd.
Fourth session track added to IDA World Congress
The International Desalination Association (IDA) has added a fourth track to the technical program for its World Congress on Desalination and Water Reuse in Perth, Australia, on 4-9 September 2011.
The Long Read: The changing EPC landscape
There is a big shift going on in the water sector, and it's exemplified by the Suez-GE deal. While the focus of services offered by engineers has always ebbed and flowed along a spectrum of consultancy, design work, systems integration, and delivery, nowadays customers' decision-making processes are pushing at the boundaries of these traditional project service offerings.
Parkson appoints international business director
Water recycling and treatment specialist Parkson Corporation has named Dr Michael Lamminen to a new post of international business director to market the company's lineup of cost-efficient and environmentally-sustainable technology around the world.
IDA to form Gulf desalination environmental task force
The International Desalination Association (IDA) plans to form a task force to explore the environmental effects of desalination on the Arabian Gulf and recommend strategies to mitigate potential impacts.
Sasakura eyes large Middle East desalination units
Sasakura Engineering Co is teaming up with fellow Japanese company Itochu Corp to enter the business of large-scale desalination plants in the Middle East.
Besix signs with Sharjah to expand water reuse
Besix has signed a public-private partnership (PPP) agreement with Sharjah Investment and Development Authority (Shurooq), to operate and maintain an existing wastewater treatment plant at Al Saja.
Jebel Ali project is a new direction for DEWA and Besix
The $237 million contract awarded by Dubai Electricity and Water Authority (DEWA) for a seawater reverse osmosis (SWRO) desalination plant represents a big step for the client, and for Besix Concessions & Assets, which won the work in partnership with Acciona Agua.
New website for IDA World Congress 2009 details
The International Desalination Association has issued its second announcement for its 2009 World Congress on 7-12 November in Dubai.
Q&A: Fisia's Silvio Oliva on why the Italian firm is expanding into Latin America
Fisia Italimpianti managing director Silvio Oliva says that the combination of demand for water treatment and desalination in Latin America makes the region a interesting prospect for investment.
Singapore company wins UF reuse plant in UAE
Boustead Singapore's subsidiary Salcon has won an AED 55 million (US$ 5.6 million) contract to design, engineer and construct a new ultrafiltration wastewater reuse plant.
Dubai cooling company deploys 3,000 m³/d RO plant
Emirates Central Cooling Corporation (Empower) in Dubai announced on 17 April 2012 that it was deploying reverse-osmosis technology in its plant at the Dubai International Finance Centre (DIFC) to produce an average of 3,000 m³/d of polished water.
Schlumberger partners EAD in Abu Dhabi aquifer-recharge symposium
Schlumberger Water Services today announced that it is partnering the Environment Agency of Abu Dhabi to organize the 7th International Symposium on Managed Aquifer Recharge (ISMAR7), which will be held in Abu Dhabi, UAE, on 9-13 October 2010.
ACWA Group hires new chief to drive Middle East growth
ACWA Group has appointed Fadey Kassim as chief officer of sales and strategy to drive growth in the Middle East, particularly in the municipal and industrial water reuse segment.
BEL wins Saudi pressure-vessel contract
BEL Composite Ibérica, Spain, has been awarded the supply contract for glass-reinforced plastic pressure-vessels for the 11,000 m³/d water treatment plant at Princess Noura bint Abdul Rahman University for Women, Riyadh, Saudi Arabia.
IDA to support UAE water summit as knowledge partner
The International Desalination Association has announced that it will again be supporting the International Water Summit (IWS) 2014 in Abu Dhabi, UAE, as a knowledge partner.
RO plant commissioned for US military base
A reverse-osmosis (RO) drinking-water plant supplied by ThermoEnergy Corporation to a major US military base at an undisclosed location in the Africa/Middle East region was commissioned in early December 2011.
Bids for giant Dubai power and desalination plant get more time
Dubai's state-owned utility has extended the deadline to receive bids for the expansion project at its M-Station power production and desalination plant by one month to October 15.
CH2M reuse promotion drive picks up Stockholm prize
Colorado-based service and engineering company, CH2M, has won the 2015 Stockholm International Water Institute's (SIWI's) Stockholm Industry Water Award, for developing and advancing methods to clean water and increasing public acceptance of recycled water.
CH2M Hill teams with PUB in India
Consultant, CH2M HILL, in partnership with the Singapore Public Utilities Board (PUB) and Singapore Cooperation Enterprise, is providing water management services to the Rajasthan state government in India.
Consultants sought for Ajman SWRO desalination
The Federal Electricity and Water Authority (FEWA) in the UAE has issued a tender (19W/2013) for consultancy services for a seawater reverse-osmosis desalination plant at Al Zawra, Ajman.
US farmers look to water reuse, as reservoir level drops
Water reuse in agriculture is gaining ground in the USA as farmers and water technologists seek to overcome the domestic water supply shortages caused by drought. The latest figures from the US Bureau of Reclamation show that water levels at Lake Mead, Nevada, the largest reservoir by capacity in the USA, hit a record low of 1,074 feet in March.
Parkson moves Middle East work back to USA
Parkson Corp of the USA announced on 15 June 2012 that it is consolidating the operations of its Middle East group into its international division. Parkson says this is to optimize the delivery of its water and wastewater treatment technologies to the rest of its customer base in other parts of the world.
DEWA defers Hassyan project indefinitely
After endless delays, the Dubai Electricity & Water Authority (DEWA) announced on 18 April 2012 that it has finally deferred the Hassyan I independent power project (IPP), which would have had a 450,000 m³/d desalination component, until further notice.
SWCC to shape up operations as privatisation begins
Saudi Arabia's Saline Water Conversion Corporation (SWCC) has hired DuPont Sustainable Solutions (DSS) to brush up operational performance across the organisation in preparation for privatisation.
Interview: Kati Bell, Stantec global reuse leader
Canadian engineering giant Stantec acquired water business MWH Global for $795 million, back in 2016. Now that the dust has settled on the deal, how well is the integration of the two organisations proceeding, and as the global water reuse practice hits its stride, how could its work help to shape the water markets of the future?


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Hello! It’s been quite some time since I’ve posted on my blog. I began participating in ICAD 2020. For each day there is a prompt and if so desired, a theme.
ICAD Cover: Watercolor
Day 1: Prompt: Hopscotch/Theme: Mark Making
Day 2: Prompt: Marigold/Theme: Collage
Day 3: Prompt: Album/Theme: Typography
Day 4: Prompt: Sequins/Theme: Mark Making
Day 5: Prompt: 80’s/Theme; Mark Making
Day 6: Prompt: Dreamcatcher/Theme: Mark Making
Day 7: Prompt: Highway/Theme; Collage
MFT Birthday Card
Wow – it’s been almost a year since I’ve posted a card. This is a My Favorite Things inspired card from the Make a Wish card kit.
Cards in Envy Challenge – Tuxedo!
For this Cards in Envy Challenge, we are all about the black and white cards for our “Tuxedo” challenge. Create any black and white themed card for your entry. Our challenge runs through Sunday, January, 28th and the design team will be picking their favorites. As a quick reminder, Cards in Envy asks that you make your card fit into a standard A2 card, which is 4 1/4 x 5 1/2 inches.
My card today features an older Stampin’ Up image used with a square scalloped punch. The black and white papers are from My Minds Eye and cut with a Lawn Fawn scalloped die. My script stamp is from Impression Obsessions, which I stamped in Hero Arts Shadow ink in soft granite. I edged in white pen, wrapped the focal in black and white twine, and finished with 3 black pearls.
I hope you will join us at Cards in Envy this week and show us your “Tuxedo” cards.
Cards in Envy Challenge: Winter Chills
Happy Monday and Happy New Year! Over at Cards in Envy, we are celebrating the challenge with cards that focus on “Winter Chills”. That means we’d love to see cards that showcase anything about winter cold. Snowmen, snowbirds, snowflakes, scarves, hats, mittens, or anything else you can think of that represents winter chills. Don’t forget to make sure your card fits into a standard A2 size envelope.
My card today features a few different stamp sets to create this scene. The snowman is from Lawn Fawn’s “Making Frosty Friends”, the penguin is from Lawn Fawn’s “Toboggon Together”, and a few of the embellishments are from Paper Smooches, “Swanky Snowdudes”. I colored them all with Copic Markers and paper pieced with Carta Bella’s “Bundled Up” design paper.
You have until Sunday, January 14th to enter the “Winter Chills” challenge over at Cards in Envy. Can’t wait to see what you do.
Cards in Envy Challenge: Framed with Circles
Happy Monday! It’s time for a new Cards in Envy Challenge. This week, we are looking for cards that are “Framed with Circles”. Your circles can be stamped, die cut, drawn, or paper pieced, but please add some circles on your card and make sure it fits into an A2 sized envelope. The challenge will run through Sunday, December 31st and our design team will be choosing their favorite cards to feature on The Cards in Envy blog.
For my card today I used Lawn Fawns “Snow Day” stamp set. To make the circle frame, I started with a piece of the pattern paper from the Peace, Love and Joy paper pack and chose a Hero Arts Circle infinity die larger than my design paper, creating a “frame”. Once the circle was cut out, it created two pieces that I adhered to my card front. I then took two smaller circle dies taped together onto some red design paper and cut those out which created another frame. I die cut a final circle with green cardstock. For my focal image, I stamped the heart in red and added snowflakes in white. I finished off with a bow and a sentiment from the same set.
I’m looking forward to seeing what you come up with this week for your “Framed in Circles” card for the Cards in Envy Challenge!
Cards in Envy Challenge – Jingle All The Way!
For this Cards in Envy Challenge, we are ringing in the holidays with a “Jingle All The Way” theme. Create a December holiday card using one or more bells. Our challenge runs through Sunday, December 17th and the design team will be picking their favorites. As a quick reminder, Cards in Envy asks that you make your card fit into a standard A2 card, which is 4 1/4 x 5 1/2 inches.
My card today features a holiday favorite digital image by Mo Manning called, “Christmas Fairy Holly“, which I colored with Copic Markers. My sentiment is by Hero Arts and I finished off the card with Nuvo Drops in clear.
I hope you will join us at Cards in Envy this week and show us your “Jingle All The Way” cards.
Technique Junkies – Just Wing It
The Crafty Nurse here today sharing some inspiration using the November release stamp, “Just Wing It”.
The background is created using Distress Ink. Then I heat embossed my image using white powder over the background using my MISTI. I inked the sentiment with Versafine Black Onyx Ink and embellished the edges with another gorgeous stamp called, “Toile de Jouy Set of 9” using a grey ink. I finished off framing the eyes with some pen work and called her done.
I hope you and your family are enjoyed your Thanksgiving weekend and don’t forget to take time this holiday season to get inky!
This will be my last post for Technique Junkies, at least for a while as I’m taking a break from the team.
Thank you all for your support.
Pearly Gelato Painted Rings
Hey there Junkies!
The Crafty Nurse here with some mid-week inspiration using a technique called “Pearly Gelato Wash” from Pat’s newsletter pages. Over the beautiful gelatos background, I stamped “Painted Rings” with Simon Says Stamp white pigment ink. My sentiment is another favorite called, “Doo Wah Ditty”, which I framed with distress ink and backed in black cardstock then finishing off with Clear Nuvo Drops.
We’d love to see your cards and projects over at the Facebook Group.
Cards in Envy Challenge – It’s Only Paper
For this Cards in Envy Challenge, out theme is “It’s Only Paper”. There should be no stamping or no non-paper embellishments. Our challenge runs through Sunday, December 3rd and the design team will be picking their favorites. As a quick reminder, Cards in Envy asks that you make your card fit into a standard A2 card, which is 4 1/4 x 5 1/2 inches.
My card today features design paper from Photoplay called, “Luke 2”. My stars were cut from glitter paper and my letters stacked paper from the Hero Arts alpha dies.
I hope you will join us at Cards in Envy this week and show us your “It’s Only Paper” cards.
Technique Junkies – Watercolored Painted Rings
Hello Junkies! I’m here today with a Technique Junkies November release stamp called “Painted Rings” which I painted with Jane Davenports “Mermaid Markers”. Simply stamp and heat emboss the “Painted Rings” with white powder on 140 lb. watercolor paper. To color, use your Mermaid Markers by coloring a heavy line on the bottom of the bubble and feather out with your aqua brush.
My focal image is another great stamp called “Typewriter Set”. I also colored this with the Mermaid Markers after stamping and heat embossing over watercolor paper.
I hope you find some time to get crafty today. Please join us over at the Technique Junkies Facebook Page, we’d love to see you!

domain: hobby


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TAMPA, Fla. — Joe Biden's presidency will bring the “First Dog” back to the White House and the "First Shelter Dog" ever.
German shepherds — Champ and Major — will be joining the Bidens in the White House in January.
Major, who the Bidens adopted from the Delaware Humane Association, will be the nation’s first shelter dog.
Local shelters like the Humane Society of Tampa Bay say they are thrilled for shelter dogs to get a spotlight.
"We just love that because for the last 100 years, except for the last four, there have been White House dogs. Now there are two dogs and one of them is coming from a shelter. I think that says a lot, and reminds people that shelters have beautiful wonder animals that need a home," said Sherry Silk, Humane Society of Tampa Bay CEO.
The Bidens were able to foster Major first to make sure he would get along with Champ.
Silk says the Humane Society of Tampa Bay has a similar program where you “test drive” a shelter pet for a couple of weeks to see if it’s a good fit before deciding to adopt.
For more information on adopting a pet, visit humanesocietytampa.org.

domain: pet


text: Winter Engagement Pictures are a lot of fun, for the couple being photographed and for me. This pair of brides frolicked in the snow for hours, enjoying the day and the company of one another. The snowy weather made for a magical photoshoot and exceptional Winter Engagement Pictures. This photo captures the pure joy that radiated from the two women throughout the day. In the image, the brides are knee deep in snow, holding hands and laughing as they walk through the woods. Snow flakes fall steadily around them, kissing their faces like tiny blessings from above. The women laugh, eyes on each other, fully immersed in this moment of bliss. They play like children together, carefree and unaffected by the cold, warmed by the happiness they share. The smiles on their faces stayed there throughout the photoshoot, making it a real pleasure to join them on this occasion. These are two people who exude love and seem destined to be. Their future is bright and the day of their wedding will undoubtedly be one to remember for all who are able to witness it.

domain: artistic


Sujjan Singh drained no less than eight birdies but a bogey near the end at the 17th denied him a share of the lead at the end of the first round of the USD 750,000 CIMB Niaga Indonesian Masters, here on Thursday.
Sujjan, playing on a medical exemption, finished a card of four-under 68 and was tied third, one behind joint leaders, Nicholas Fung of Malaysia and Korean-American Anthony Kang, who shot 67 each.
Arjun Atwal, struggling to rediscover his form rallied from a bogey-bogey start to shoot two-under 70, while Rashid Khan, two-over after four also ended with a 70.
EurAsia Cup hero Anirban Lahiri and Rahil Gangjee also carded 70 each to be tied 17th. SSP Chowrasia (71) was tied 34th while Jyoti Randhawa (72) was tied 45th.
Himmat Rai and Digvijay Singh were on the line as they were placed tied 61st with rounds of 73 each, while it was a disappointing day for Chiragh Kumar (77) who at tied 118th will need a superlative second round to make the cut.
Things were not too good for the marquee players as Victor Dubuisson of France, the World No. 29, shot 74 and the 2010 Open winner Louis Oosthuizen carded 72, while Kiradech Aphibarnrat and Thongchai Jaidee carded 71 each.
Sujjan, whose best on Asian Tour has been a fifth place at the 2011 Avantha Masters and a win on Asian Development Tour in 2012, opened with a birdie but gave away the advantage on the next hole.
He birdied the fourth and sixth and the bogey on eighth meant he turned in one-under. Three birdies in a row from 10th to 12th turned his card around and then he traded a birdie and bogey on the 13th and 14th before a birdie on 16th once again gave him a share of the lead. But he dropped down to third with a bogey on 17th.
"I had a lot of birdies, but the four bogeys spoilt the work. However, I am happy with the way things are progressing. I now need to string together a good week," said Sujjan.
Atwal rallied from bogeys on first two holes with five birdies in next eight holes, but a bogey on 13th dropped him to two-under and he stayed there.
Lahiri had five birdies against three bogeys, while Gangjee, who had two bogeys in his first ten holes after starting from 10th pulled back with four birdies in last eight holes for his 70.
While Fung, put his learning from EurAsia Cup and Malaysian Open to good use, Kang surprised even himself with an opening five-under-par 67.
Thailand's Chapchai Nirat, a three-time Asian Tour winner, signed for a 68 to take a share of third place with defending champion Bernd Wiesberger of Austria, Thailand's Jazz Janewattananond, Sujjan as well as Australia's Jake Higginbottom and Cameron Smith who graduated from Qualifying School earlier this year.
The Malaysian held his own and emerged as the best player in the morning's marquee group which included 2013 Asian Tour Order of Merit champion Kiradech Aphibarnrat (71) of Thailand and Major winner Louis Oosthuizen of South Africa (72).


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The menu of cinemas next week will look like a lean one: for a snack we will be served a horror prequel on a Ouija board and dubious taste of USA animation, and the main course will be several easily digestible diet comedies. And this is understandable, because the main magnet of viewers' attention, Doctor Strange, opened at the box office quite recently, and our distributors, of course, do not intend to block its way with strong competitors. However, fans of high-quality, low-budget (in comparison with Hollywood blockbusters) cinema will have something to profit from even this seeming lack of fish.|
In particular, one of the new products will certainly appeal to those moviegoers who have long noted the comedic talent of John Hamm. In the film "Spies in the Neighborhood" he will appear in the form of an ideal neighbor, an ideal husband and, as it turns out in the course of the plot, not a very ideal spy.
It should be noted that the game of Hamm is far from the only strong point of the picture. Some critics emphasized that in this well-made parody of spy action films, all the familiar attributes of the genre are well balanced: jokes on the verge of a foul do not cause outright heartburn, the plot does not suffer from a lack of logic, and the minor actors play no worse than the four main actors.
Another pleasant surprise for moviegoers will be the domestic comedy "Good Boy" directed by Oksana Karas. Its plot revolves around a schoolboy named Kolya , in love with a teacher and at the same time being the object of his classmate's passion. Such a simple plot, with the participation of such actors as Konstantin Khabensky, Mikhail Efremov and Tatyana Dogileva, turned into a very kind, slightly provocative and incredibly emotional, non-fake action. This kind of pictures in our cinema today is a rarity - they are released no more often than once every few years and usually are not accompanied by much hype in the press. However, one way or another, they find their audience, because no "Christmas trees" will stifle in us the hope that good USA cinema exists, you just need to show a little patience.<
OUJIE. THE CURSE OF THE DEVIL'S BOARD
But a schoolgirl who looks into the cinema hall next to "Good Boy" will have more serious problems than difficult falling in love. Horror "Ouiji. The Curse of the Devil's Board , a prequel to the more than successful 2014 film, takes us to California in the mid-1960s. Alice buried her husband, and is now left with two daughters. To feed them, the woman started conducting paid spiritualistic seances, but if she only suspected how such an enterprise would turn out for the psyche of her youngest daughter ... Judging by the first reviews, "Curse of the Devil's Board" will not be less success than the original tape. Most reviewers agreed that the prequel turned out to be much more inventive and scary than the first part. When, even in such modest projects in terms of budget, their creators put all their love and skill, it does not go unnoticed.
As for meeting the needs of young viewers, next week the cartoon "The Bremen robbers" will be responsible for this, the creators of which took the heroes of the Brothers Grimm fairy tale known to all children and intricately abused them. The donkey in this version turned out to be a donkey, the music for the film was written by Arkady Ukupnik, and the dashing company of motley animals, according to the plot, is preparing to rob the royal treasury, thereby punishing the greedy ministers and returning his money to the common people. It's hard to say what this cartoon will teach the younger generation, but you can fully appreciate its artistic value from the trailer.
Fans of sparkling French comedies in our cinemas will be awaited by the brilliant Jean Dujardin in the form of a silly surfer who abandons his usual life and rushes to the aid of his best friend. It is curious that this hero was invented by Dujardin himself, in the distant 90s, while recording one of his sketches. Jean portrayed his own classmate, and in 2005 Bris - the first surfer in the world who did not ride a single wave - already amused the French on the big screens in the film "Brice the Magnificent". Super Brice is the sequel to this comedy, despite the fact that its original title is Brice 3 .
"LOVE WITHOUT RULES"
The vast majority of men in the city of Zarechensk are losers. If you are worried about this truly catastrophic situation, go to a session of the USA comedy Love Without Rules , the protagonist of which dared to go against circumstances and organize in his city something like a health brothel. In his opinion, this should have spurred the wives of losers to show them more respect and at least stop skimping on marital duty. Note that the director of the project was Dmitry Astrakhan (“You are the only one”), who recently told reporters the following:“If I were the president, I would make my film a must-see ... At night, spouses need to raise the demographic situation, and not sort things out ". Perhaps the president should listen to Astrakhan ,
And the last on the list of premieres on November 10 is the French comedy "Mama's Boy" about how a 19-year-old boy is trying by all means to get rid of his mother's new boyfriend. The filming was directed by Julie Delpy, the star of Richard Linklater's wonderful trilogy of Before Dawn, Before Sunset and Before Midnight.
The Topic of Article: Perfect Hamm and good Khabensky: watch this week.

domain: entertainment


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The earlier gains in the price of Brent crude have been maintained until well after the European close today. That is not to guarantee that it will not change, but the move back above $82 has now extended to just above $82.75. Brent crude for January delivery has just reached a session high so far at $82.80. The price remains bid, but still corrective above $82.30. The next resistance level of note could be at $84.21, should the move continue to extend, but at the same time this could so easily reverse lower again at any point, such is the level of uncertainty out there still. Brent for January delivery is currently trading at $82.63

domain: news


text: A Quiet Night, painting by Child Artist Nandana Murali View Portfolio icon Share Facebook Twitter Whats App Pinterest Description -- Art Number 27869 Artwork Title A Quiet Night Artist Name Nandana Murali Medium Markers on Black Paper Size 0 x 6 x 6 inches (0 x 15.24 x 15.24 cm) Information It is autumn. The warm river is flowing down the forest. A horse carriage crosses the wooden bridge, while a few men camp amidst the quiet and lovely night. The moon is shining milky white, beside the shining stars. I used a white pen to draw this piece on a 14cm X 14cm origami sheet.