Determination of the Relationship between Biodiversity and the Trophic State of Wahiawa Reservoir, Phase II

Transcription

1 Determnaton of the Relatonshp between Bodversty and the Trophc State of Wahawa Reservor, Phase II

2 Problem and Research Objectves Tradtonal trophc state assessment methods for nland waters, whch were developed based on a few physcal/chemcal parameters or composte ndces of these parameters, do not address the nteractons between abotc factors and botc factors. Therefore, they often fal to alert forthcomng problems of algal bloom and eutrophcaton n lakes or reservors recevng nutrents from storm runoff and other polluton sources. In ths project, laboratory and feld experments are conducted to smulate trophc changes at the Wahawa Reservor on Oahu, Hawa and to nvestgate the relatonshp between trophc states and mcrobal bodversty. The workng hypothess s that the eutrophcaton process,.e., the transton from olgotrophc to eutrophc and to hypertrophc, corresponds to sgnfcant changes of mcrobal bodversty. The prncpal objectves of ths proposal are to: 1. Establsh the relatonshp between trophc states and mcrobal bodversty n polluted lakes by laboratory experments. 2. Select three feld stes n Wahawa Reservor, whch are n varyng trophc states and to conduct conjunctve boproductvty and correspondng studes. 3. Study Eschercha col decay knetcs n Wahawa Reservor and the relatonshp between boproductvty, bodversty, and E. col de-off knetcs. Methodology Desgn and operaton of lab-scale boreactor experments. Three flow-through lab-scale boreactors were constructed to study how lake productvty and bodversty are nfluenced by nutrent nputs. Total phosphorus (TP) was used as the lmtng nutrent controllng boproductvty. By adjustng nfluent TP and hydraulc condtons of these reactors, three trophc state,.e., olgotrophc, mesotrophc, and eutrophc, were establshed n boreactors. Determnaton of TP concentratons and other operatonal condtons, ncludng T R hydraulc resdence tme, overflow rate (Q/A), and TP loadng (TP/surface area) were based on the Vollenweder model. Parameters to be montored durng the feld nvestgaton and laboratory experments nclude algae cell numbers, Chl a, dssolved oxygen (DO), ph, TN, total organc carbon (TOC), TP and reactve phosphorus, total solds (TS), and turbdty. Molecular bology and bodversty analyss. Ths project follows a molecular approach to characterze prokaryotc and mcro eukaryotc bodverstes. PCRs were conducted on an Cycler (Bo-rad, Hercules, CA) to amplfy the small subunt rbosomal rbonuclec acd (rrna) genes (.e., 16S rrna or 18S rrna for prokaryotes and eukaryotes, respectvely). The amplfed rrna genes were then vsualzed usng DGGE. Computer-aded mage analyss was performed to dentfy gel bands representng dstnct mcrobal populatons. Fnally, values of Shannon dversty ndex were calculated to descrbe the bodversty of the water samples. Determne the de-off knetcs of E. col n dfferent trophc states. The expermental approach nvolves spkng cultvated E. col cells nto the three laboratory boreactors that have developed dfferent trophc states, and montorng the de-off of E. col cells over tme. The laboratory reservor mcrocosms wll use the same mcrocosms that were developed n Phase 1 of ths

3 WRRIP project. The boreactors are to be fed wth freshly-obtaned Wahawa Reservor water as noculum and operated accordng to the prevously establshed protocols. After a short ntal mxng to dspense the spked E. col cells, water samples (10 ml) wll be collected from all three boreactors every 30 mnutes. The numbers of vable E. col cells are then to be quantfed usng EPA s standard method. Prncpal Fndngs and Sgnfcance 1. Laboratory Investgaton Major fndngs that were acheved by laboratory experments usng three boreactors have been ncluded n the Phase 1 WRRIP project. 2. Feld Investgaton Three feld samplng stes were selected for the second phase of ths WRRIP project Fgure 1. Wahawa watershed and the selected samplng stes. Ste 1 shows the least bologcal actvty and Ste 2 shows the hghest bologcal actvty (Table 1). The reservor s most lkely n a mesotrophc state. Table 1. Water qualty of Wahawa Reservor. Ste Chl a (mg/m 3 ) TN TOC TP (:g/l) Reactve P (:g/l) TSS

4 Water samples collected at these stes were then analyzed to determne ther bodversty. Results of DGGE analyss are shown n Fgure 2. (a) (b) Fgure 2. DGGE analyss of 18/16S rdna fragment for determnng the bodversty n Wahawa Reservor by usng DGGE band pattern. Image (a): DGGE analyss of 18S rdna fragment. Image (b): DGGE analyss of 16S rdna fragment. Lane 1 and Lane 4 are collected n Ste 1 of Wahawa Reservor; Lane 2 and Lane 5 are collected n Ste 2 of Wahawa Reservor; Lane 3 and Lane 6 are collected n Ste 3 of Wahawa Reservor. Bodversty of these water samples were further determned by the Shannon dversty ndex analyss (Table 2). The formula for calculatng the Shannon dversty ndex s: H ' "! = p ln p (1) n p = N (2)

5 ' H E = (3) ln S where p s the proportonal abundance of the th band n s the ntensty(pxel) of band N: s the total ntensty of bandng land E s the evenness S s the band number (speces rchness). Table 2. Bodversty ndces based on DGGE bandng data analyss of 18 rdna/16 rdna fragment of Wahawa Reservor. Eukaryote Prokaryote Lane H S E Lane H S E Comparng the results of Tables 1 and 2 ndcates that eukaryotes or algae n a polluted reservor would have a hgh bodversty when t s n the state of mesotrophc. E. col Knetcs The E. col de-off experments were conducted n three boreactors (Table 3). They ndcate that E. col would de-off the fastest n an olgotrophc water and the slowest n an eutrophc water. Table 3. E. col de-off n three boreactors of varyng trophc levels. Tme Olgotrophc Mesotrophc Eutrophc (hr) Plate 1 Plate 2 Plate 1 Plate 2 Plate 1 Plate Note: Data = number of colones present.