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SOME CHEMICAL AND MICROBIOLOGICAL OBSERVATIONS IN THE PACIFIC OCEAN OFF THE HAWAIIAN ISLANDS K. Gundersen, C. W. Mountain, Diane Taylor, R. Ohye, and J. Shen Department of Microbiology, University of Hawaii, Honolulu 96822 ABSTRACT Simultaneous chemical and microbial analyses of the water column were made at 33 stations off the leeward Hawaiian Islands. The overall distribution of oxygen, nitrate, and pH was similar at all stations located more than 5 km offshore. These parameters were closely correlated and also correlated with nitrifying and nitrate-reducing activity in the water column. The distribution of nitrite and ammonium did not correlate with the oxygen distribution. A nitrite band was consistently found in the lower portion of the photic zone and appeared to have originated from reduction of nitrate rather than from oxidation of ammonium. The distribution of aerobic and anaerobic bacteria was regulated by the amount of available organic nutrients and not by the oxygen concentration. INTRODUCTION Little information is available regarding the water chemistry and microbiology off the leeward Hawaiian Islands, Earlier microbiological work (Adair and Gundcrsen 1969a, b; Ohyc and Gundersen 1969) was not accompanied by chemical-physical analyses of the ocean environment, and we were therefore not in a position to draw any conclusions as to the ecological significance of our findings. We became convinced, then, that to understand microbial processes as they occur in the sea it is imperative to make simultaneously precise analyses of biologically important parameters of the water column and to relate these to the processes studied; during subsequent cruises such parameters were routinely determined along with microbial analysis. During 1969, we made 17 separate cruises (Bug Scafari) off the leeward Hawaiian Islands, from Kawaihae Bay, Hawaii, in the east to Niihau in the west (Fig, 1). Altogether 33 stations were investigated, 14 of which were on or beyond the l,OOO-fathom contour. No station was closer to land than 5 km. Station 17A was 1 Hawaii No. 398. LIMNOLOGY Institute AND of Geophysics OCEANOGRAPHY Contribution revisited twice in early 1970 and some of the observations made during these later cruises have been included in this report. Northeast winds, rarely exceeding 10 m /set, were prevailing during the cruises. All cruises were made on the RV Teritzc. We are indebted to Mr. L. I. Knowles and to officers and crew of the RV Teritu for their interest and cooperation during this work. METHODS Sampling Water samples for most chemical analysis, salinity, and pH were taken with a set of twelve 13-liter PVC samplers (a locally manufactured modification of the Van Dorn sampler); samples for particulate organic matter and protein were taken with Niskin sterile bag samplers (General Oceanics, Inc. ) ; bacterial samples were also obtained with these samplers and with JZ sterile bottle samplers (Kahl Sci. Instr. Corp. ) . Samplings were made at 50-m intervals, 12 bottles to the cast; in the upper water column, closer intervals were sampled in several cases. The reported depths are taken from the winch meter and corrected, when rcquircd, for wire angle. 524 JULY 1972, V. 17(4) ANALYSES 160” -- 159” OF IIAWAIIAN OCEAN 158” WATIZRS 157” 156” l55”W L,I-- 22”N IOA l ,’ I.-_. HAWAIIAN ISLANDS -.. -- --.--.--+ FIG. 1. Bug Seafari cruises, 1969. Physical-chemical methods The temperature of surface water, taken in a bucket, was recorded to the nearest 0.5C. The temperature profile of the upper 275 m was determined on every station with a standard bathythermograph. Equipment was not available for temperature measurements below this depth. Light penetration was measured with a locally manufactured light transmission metering system, having a range from 3001,000 nm and with a sensitivity from 107107,640 lux. Samples for salinity were stored in tightly stoppcred glass bottles and analyzed in the labloratory with a Hytech salinometer (model 6210). pH and alkalinity were dcterrnined with a Beckman pH meter according to standard procedures I(Strickland and Parsons 1968). Because temperature data were not available from depths belomw the reach of the bathythermograph, we were not able Position of stations. to make tempcraturc corrections for the pH readings 1 -~ .y so the. values . . reported are Lower than the true in situ values. Oxygen was determined routinely by two independent methods : polarographically with a Beckman oxygen analyzer (model 777) and chemically by standard Winkler titration. The two methods showed good correlation but only the values obtained by titration are reported here. Ammonium was determined according to the method of Solorzano (1969). Nitrite, nitrate, and reactive phosphate were determined according to methods described by Strickland and Parsoas ( 1968) . Samples for particulate protein analysis were filtered through 47-mm Millipore HA filters (pore size, 0.45 p). The amount of water varied with the origin of the samplc: 800 ml of surface water were filtered; 1 liter from between 25 and 500 m; 1.5 liters between 500 and 1,000 m; 2 liters below 1,000 m. The filters were trcatcd 526 K. GUNDERSEN according to the Lowry-phenol method for protein analysis ( Lowry et al. 1951). A 15-min alkaline hydrolysis was used, followed by the addition of Folin’s reagent. The absorbance was read after exactly 30 min at 750 nm. Water for particulate carbon (POC) and nitrogen (PON) analyses was transferred to e-liter polyethylene bottles which had previously been rinsed with distilled water made particle-free by filtration through 0.22-p Millipore filters. The actual filtering was through 1.2-p Sclas Flotronic silver membrane filters (45 mm) in a Millipore Pyrex filtering assembly under a vacuum of less than 550 mm of mercury. One-liter quantities wcrc filtcrcd and the filtering funnel washed with particulatcfree distilled water bctwecn filtrations. The filters were individually stored in pctri dishes until analyzed in a CHN analyzer (Hewlett-Packard F&M, model 185) according to standard procedure. Unless otherwise stated, all chemical analyses were made on shipboard. Microbiological methods Standard Milliporc filtration of asep titally sampled water was used to determine bacterial distribution in the water column. Samples of lo-100 ml were passed through 47-mm Milliporc IIA filters (porosity, 0.45 p) using a manifold and a vacuum of about 700 mm of mercury. Four or six filtrations were made of each sample and the filters subsequently placed on marine agar (Difco) plates. Half the plates were incubated aerobically in the dark and the other half incubated under hydrogen gas in anaerobic jars (Torsion Balance Co.). Plates were examined after 48-72 hr of incubation at 28 rt 2C. Most aquatic bacteria occur in aggregates of variable numbers of individual cells and in close spatial proximity on the surfaces of particulate matter (ZoBcll 1946; Scki 1970) and consequently give rise to considerably fewer colonies than there were cells in the original sample, so we have preferred to report bacterial counts as “colony-forming ET AL. units” ( c.f.u. ) , rather than as “number of cells,” per unit volume. Several different tcchniqucs wcrc used in the investigation of nitrification and nitrifying microorganisms in the water column but only a few will bc reported here; a more cxtensivc report of methods used in nitrogen transformation studies in these waters was recently given by Mountain ( 1971). Enrichment cultures were prcpared by injecting a few milliliters of ( NH4)&04 or NaN02 solutions directly into the polycthylcnc sampling bags, cstimated to give final concentrations of about 1,000 pg-atom N/liter. Exact detcrmination of initial ammonium, nitrite, and nitrate concentrations was made on subsamples withdrawn immediately after cnrichmcnt. During the incubation period of several weeks in the dark at 25 -L 2C, water was withdrawn at intervals for chcmical and bacteriological analysis. The same tcchniquc used for nitrification was used for nitrate reduction. Enrichments consisted of NaN03 (about 1,000 pug-atom N/liter) alone or in combination with glycerol ( 0.1% ) . All the initial microbiological work was done without delay on shipboard. RESULTS AND DISCUSSION The overall vertical distribution of pH, oxygen, nitrate, phosphate, protein, particulatc organic matter, and bacteria in the water column was within the cxpccted range for the pelagic distribution of these parameters. A slight, but consistent oxygen maximum was found near 100 m and a minimum, of about 1 mg Oa/liter, cxistcd bctwecn 700 and 900 m (Fig. 2A). The salinity data agree well with data given by Wyrtki ct al. (1967) and Scckcl (1968) and show a sharp maximum near 100 m ( Fig. 2B), characteristic of North Pacific Central Water. Below this water mass and down to the lower depth of this study (1,150 m), the low salinity North Pacific Interrnediate Water makes up the water column. Whereas nitrate was practically absent from surface waters at all times, a fairly ANALYSES A. IO 15 20 OF IIAWAIIAN OCEAN 527 WATERS 7.50 175 8.00 8.25 pH 25 30°C T c.f.u.bacteria/titer \ b ‘Aerobic bacteria Anaerobic I: CO% 1.90 780 PH R -z c. 20 25 .50°c 1.95 8.00 2.00 mM/li ter 8.20 T 15.0 20.0 25.O”C T 0 66 cm0 2 4 6 8 IO ,uq PON-N/liter 0 20 40 60 80 IOOjkj POC- C/liter 0 1 10.0 I% \iqht penet 8.00 8.25 pH I bacteria 2 4 6 I 8 x 104c.f.u. bacteria/liter I I I IA- p.m. FIG. 2. Characteristics of the water cohmm of three stations investigated during the Bug Seafari cruises off the leeward Hawaiian Islands. A. Station 6B, off Oahu. pH uncorrected. Bacterial counts are reported as colony-forming units (c.f.u.). B. Characteristics of the upper 300 m of station 17A, off Oahu. C. Station 5B, a shallow nearshore station off Maui. The two casts were made at 0800 and 1600 hours. 528 K. GUNDERSEN sharp nitrite band, amounting to 0.06-0.07 pug-atom N/liter, was consistently found between 100 and 200 m ( Fig. 2B). There was, however, no nitrite band associated with the oxygen minimum at about 800 m. Ammonium was most abundant in the upper water column but rarely cxcceded 0.5 pg-atom N/liter. Its distribution was usually irregular and did not always show a clear peak ( Fig. 2B ) . In deeper water, nitrite and ammonium were usually absent, but nitrate increased rapidly below about 150 m reaching a high of about 40 pug-atom N/liter at about 800 m. The highest value measured was about 47 pg-atom N/liter below 1,000 m. Minor fluctuations in the chemical paramctcrs over the year apparently were not seasonal variations. The temperature of the surface water varied about 5C betwecn winter and summer. The island effect described by Doty and Oguri (1956) was not reflected in our chemical data. For example, there was little diffcrcnce in the three stations 6A, 6B, and 6C, situated along a track perpcndicular to the Waianae coast of Oahu. In the shallow waters off Maui (station 5B, Fig. 2C) and between Molokai and Lanai (station 1OD) there was no significant vertical variation of any chemical paramcter and little, if any, numerical differcnce in data from a comparable depth farther offshore. However, bacteria were 4-5 times more abundant in the shallow ncarshore water than in the open ocean. One parameter possibly reflecting a land influence was the vertical distribution of particulate matter. The values for POC and PON were more irregular and usually also higher than at the windward station Gollum (22” 10 N, 158” 00 W) investigated by Gordon ( 1970). Since we used exactly the same tcchniquc and equipment, the data are comparable. The higher values and the irregular distribution on the leeward side of the islands may be due to the dumping into the ocean of large amounts of solid wastes from Hawaiian sugar mills; windrows of such materials were frequently observed during the ET AL. TABLE 1. Particulate organic carbon (POC) and nitrogen (PON), Bug Seafari cruises, 1969, station 1 SD Depth (ml O-5 50 100 150 250 300 400 450 500 550 600 650 700 750 800 900 950 1,000 1,050 1,100 POC PON ( m/liter) 62.17 32.71 68.46 21.36 27.43 26.59 18.04 59.29 43.02 50.66 45.08 45.09 15.86 22.85 37.29 26.29 30.41 55.40 21.93 13.87 10.30 6.57 11.53 7.10 5.85 6.01 5.07 9.38 8.53 5.03 6.07 6.57 4.94 4.16 6.97 3.76 2.59 8.60 3.70 3.66 C:N (w/w) 6.0 5.0 6.0 3.0 4.7 4.4 3.6 z-i 10:1 7.4 6.9 z*z 514 7.0 11.7 6.4 6.0 3.8 cruises, sometimes far out to sea. These particles continuously settle throughout the area and the prcsencc of such wastes should easily be detected in the POC-PON analysis. The high C : N ratios occasionally found ( Table 1) would tend to indicate a predominance of particulate matter of plant origin such as cellulose and other polysaccharides . The C : N ratio of whole mixed plankton is about 5.7 on a weight basis (Fleming 1940). Carbon-nitrogen analyses made on whole plankton collected in a 60-p-mesh net at about 5-m depth during one of our cruises gave C :N ratios between 4.65 and 5.20. Since the bulk of particulate organic matter in the sea originates from plankton, a high C : N ratio could also mean that a fraction of the nitrogen-containing components of dead plankton had already been decomposed in the upper portions of the water column, liberated as ammonium, and rapidly reassimilated by the phytoplankton, Our nitrification studies support this belief. As was the case with POC and PON, the distribution of particulate protein and ANALYSES Nitrate oo g&lo900- in W~~WCO~WWI 5 10 15 0 0 % E IOOO- 2,o l 00 l 25 l e 0 IIAWAIIAN jKptorn NO,-N/liter 30 35 4 : l l :. 0 P 1100 - 0 1200 I I I -20 -15 -10 -5 0 Chanqe in nitrate durinq incubation OF I 5 l I I IO 15 : pq-atom NO,-N/liter FIG. 3. Nitrifying activity and nitrate reduction demonstrated by changes in nitrate concentration of water samples during 30 days of incubation at room temperature in the dark. *No enrichment; O-enriched with 1,000 pug-atom N/liter as (NH4)2SOe. The water samples were collected and subsequently incubated in presterilized Niskin polyethylene bags. bacteria showed considcrablc vertical variation. Most of the: protein was found in the upper portions of the water column, as expected, but occasionally substantial values were found as deep as 500 m. Below this depth the values were always low. A remarkably good correlation was found bctwecn the distribution of particulate protein and bacteria when these paramctcrs were determined simultaneously (Fig. 2A). The filtration method used for protein analysis will, of course, include almost all the bacterial protein of the sample, and 30-50% of the protein measured by this method is of bacterial origin. The pattern of vertical distribution of OCEAN 529 WATERS bacteria and protein indicates the existcnce of narrow biotic strata in the water column. We have found such bands on several occasions, and they do not appear to bc technical artifacts. Sorokin (1971) described similar layers of plankton and bacteria in tropical waters. Nitrification was only detectable below 200 m (Table 2, Fig. 3). Although ammonium was produced in samples of unenriched surface water incubated fo,r 30 days in the dark, it was not nitrified (Table 2). Apparently, nitrifying organisms are either absent, or present in low numbers, in surface waters; Carlucci and Strickland (1968) estimated their numbers to be less than one cell per liter in the open ocean. A possible explanation for their insignificant role could bc the inability of the thermodynamically disadvantaged nitrifiers to compete with the phytoplankton and heterotrophic microorganisms for the small amount of available ammonium. In deeper water, however, the absence of competing phytoplankton and less o,rganic matter to support heterotrophic growth ,would favor the nitrifying organisms. Consistent with this point of view is the nitrifying capacity of most samples taken from below about 200 m ( Fig. 3). Bcforh the results of this experiment can be intebrctcd, it is necessary to realize that two apparently opposite biological proccsscb, nitrification and nitrate rcduction, both occur in the water. The factors controllibg nitrification are the availability of ammonium ( as an energy source), car- 2. Changes in inorganic nitrogen (in ,ug-atom N/liter3 in unenriched water samples from the upper 300 m of the water column following 30 days of dark incubation at 26C. Samples were taken and incubated in sterile Niskin sampler bags. Bug Syfari cruises, 1969, station 17A TABLE Initial Depth (111) NH,+ NO,- 100 150 200 250 300 0.14 0.24 0.50 0.52 0.14 0.00 0.00 0.02 0.01 0.02 0.09 0.03 0.02 0.02 After 30 days NO,- NH,+ NO,- 0.42 0.50 0.36 1.78 4.80 9.60 16.00 4.82 6.67 9.27 10.01 2.98 1.35 1.25 0.11 0.09 0.13 0.19 0.20 0.45 0.58 Increase WI- NE&+ NO,- NO,- 0.62 0.62 0.56 1.88 4.93 11.40 20.00 4.68 6.43 8.77 9.49 2.84 1.35 1.25 0.09 0.08 0.11 0.10 0.17 0.43 0.56 0.20 0.12 0.20 0.10 0.13 1.80 4.00 530 K. GUNDERSEN bon dioxide (as a carbon source), and oxygen ( as an electron acceptor ) , thus : NH4’ + 02 + COz --) Cells + NOS-. (1) Nitrate reduction, on the other hand, is controlled by the availability of organic matter (as a carbon and energy source) and nitrate (as an electron acceptor), thus: Organic matter + NOJ- + Cells + NOz- (2) (other forms of reduced nitrogen, e.g. NZ, may bc the end product), Facultative anaerobic bacteria which reduce nitrate arc present at all depths in the sea and nitrite production from nitrate, even in the prcsencc of oxygen, can easily bc dcmonstratcd in water samples enriched with glycerol or other organic matter (Mountain 1971). Thus, with only small amounts of ammonium available to the nitrificrs, nitrate reduction will be the predominant process and any nitrification bccomcs masked. On the other hand, if ammonium is added and nitrifying organisms are prcsent, nitrification becomes predominant and will mask nitrate reduction, In the unenriched water bags (Fig. 3, open circles ), the preexisting nitrate was rcduccd to nitrite whereas in the ammonium-enriched bags ( solid circles ) additional nitrate was formed. Nitrite accumulated only as a result of nitrate reduction and not as a result of oxidation of ammonium by nitrifiers. If it can bc assumed that nitrate reduction will occur whether or not there is simultaneous nitrification, the nitrifying capacity of the enriched watcr samples in Fig. 3 must bc considerably greater than shown. We had expected to find some corrclation bctwecn the distribution of oxygen in the water column and the distribution of aerobic and anaerobic bacteria, but this was not the cast. Although oxygen distribution is apparently corrclatcd with the distribution of aerobic bacteria, it is also correlated with the distribution of bacteria capable of growing in the complctc absence of oxygen, at least below the photic zone (Fig. 2A). The finding that about ET AL. two-thirds of all heterotrophic marine bactcria isolated by standard techniques, irrespcctivc of what depth and oxygen level they came from, will grow whether oxygen is available or not (Ohyc and Gundersen 1969) seems to support the view that oxygcn within the range found in oceanic waters dots not have a significant controlling cffcct on the distribution and biochemical activities of marine bacteria (ZoBcll 1940). Even the aerobic marinc nitrifying bacterium Nitrosocystis oceanus will oxidize ammonium to nitrite at low oxygen tensions ( Gundcrsen 1966; Carlucci and McNally 1969). Th e very close correlation between the distribution of protein and of heterotrophic bacteria (Fig. 2A) supports the view that the distribution of nutrients plays a much more important role in the distribution of bacteria than dots oxygen. REFERENCES 1969a. ADAIR, F. W., AND K. GUNDERSEN. Chemoautotrophic sulfur bacteria in the ma1. Can. J. Microbial. 15: rinc environment. 345353. 1969h. Chemoautotrophic AND -. -7 sulfur bacteria from the marinc environment. 2. Can, J. Microbial. 15: 355-359. GAIILUCCI, A. F., AND P. M. MCNALLY. 1969. Nitrification by marine bacteria in low conLimcentrations of substrate and oxygen. nol. Oceanogr. 14: 736-739. AND J, D. H. STRICKLAND. 1968. The iso?lation, purification and some kinetic studies of marine nitrifying bacteria. J. Exp. Mar. Riol. Ecol. 2: 156-166. DOTY, M. S., AND M. OGURI. 1956. The island mass effect. J. Cons., Cons. Perm. Int. Explor. Mer 22: 33-37. FLEMING, R. II. 1940. 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