Cyclical changes in biomass productivity and amino acid content of freshwater macroalgae following nitrogen manipulation

Algal Research 12 (2015) 477–486

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Cyclical changes in biomass productivity and amino acid content of freshwater macroalgae following nitrogen manipulation Andrew J. Cole ⁎, Alex R. Angell, Rocky de Nys, Nicholas A. Paul MACRO — the Centre for Macroalgal Resources and Biotechnology, College of Marine and Environmental Sciences, James Cook University, Queensland 4811, Australia

a r t i c l e

i n f o

Article history: Received 20 May 2015 Received in revised form 29 September 2015 Accepted 12 October 2015 Available online xxxx Keywords: Starvation Oedogonium Protein recovery PAM Photosynthetic capacity Aquaculture

a b s t r a c t The effective supply of nitrogen to algal cultures is an important aspect of intensive cultivation and critical if the biomass is to be used as a source of protein. In this study two complementary experiments examine how variation in the supply of nitrogen to cultures influenced the biomass productivity and protein content of the freshwater macroalga, Oedogonium. The first examined how robust Oedogonium is to the intermittent supply of nitrogen (supplied weekly, every second week or every third week) by quantifying its biomass productivity, photosynthetic capacity and internal nitrogen content through time. Biomass productivity over a 12-week period was highest (10.6 g DW m−2 day−1) when nitrogen was supplied weekly and lowest (8.1 g DW m−2 day−1) when nitrogen was supplied every third week. The second experiment examined the recovery of nitrogen and amino acids in the biomass following periods of nitrogen-depletion. Prolonged periods (2 weeks) without nitrogen reduced the internal nitrogen and amino acid content of the biomass by up to 80%. However, in all treatments the internal nitrogen content recovered within 24–48 h and the amino acid content had recovered within 72 h following the resupply of external nitrogen. These results demonstrate that nitrogen should be supplied in a relatively constant manner to maximize the growth rates of Oedogonium; however, the protein in nitrogen-deplete cultures can be rapidly rejuvenated by the addition of nitrogen in the days prior to harvest. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The annual consumption of protein by domesticated animals exceeds 150 million tonnes and this consumption is expected to double by 2050 [10,51]. Currently there is limited capacity to increase the production of traditional protein sources such as legumes and cereals as these crops are restricted to arable, fertile land, and require large quantities of synthetic fertilisers, many of which are finite resources themselves [21,32,47,49,50]. To overcome this future discrepancy in supply and demand, unconventional sources of protein will be required [2,10, 52]. Freshwater macroalgae are a promising candidate to meet this demand as they have several advantages over existing protein crops, including the capacity to be cultivated on non-arable land and to utilize waste sources of water, nitrogen and phosphorous [15,16,26,39,54]. Under these conditions macroalgae are also capable of very high rates of biomass production, such that on an areal basis the quantity of protein produced can be up to ten times that of soybeans [16,35], which are currently the most widely used source of protein for animal feeds [10]. Furthermore, the integration of macroalgal cultivation within existing animal production industries can create a closed loop process, through which the macroalgae assimilate and convert the waste sources of nitrogen from intensive animal production into a high quality source ⁎ Corresponding author. E-mail address: [email protected] (A.J. Cole).

http://dx.doi.org/10.1016/j.algal.2015.10.010 2211-9264/© 2015 Elsevier B.V. All rights reserved.

of protein [16,54] and this macroalgae can then be used as a feed ingredient in the diets of livestock animals [18,33]. The limiting factor in the development of any new application from algae (either macroalgae or microalgae) is the reliable, intensive cultivation of biomass at large scales [20,23,35]. At the commercial, multihectare scale the supply of water and nutrients, primarily nitrogen, are critical to biomass productivity and the ability to balance the delivery of nutrients with the requirements for algal growth will be a major challenge [13]. In places where wastewater is rich in nutrients, one possible solution is to deliver nutrients through discrete pulses, such that the nutrients in the wastewater are effectively utilized and not lost in discharge, which occurs if cultures are maintained under constant flow [15,39,54]. A useful biological characteristic of both freshwater and marine macroalgae, which may dampen any negative effects of the intermittent supply of nutrients, is the high level of plasticity in their internal nitrogen content [5,16,39]. The internal nitrogen content of algal biomass ranges between 0.8–6.9% depending upon the amount of nitrogen available in the external environment [15,16,26,39,41] and some species can maintain high rates of growth in the short term when nitrogen supply is limited [12,27,41,45]. For example, the marine macroalga Gracilaria tikvahiae can maintain its growth rate for two weeks using nitrogen reserves. Moreover, the internal reserves of this species are rapidly recovered when nitrogen is re-supplied, enabling high rates of growth to be maintained [45]. In a similar manner, the fastest growth rates of the freshwater macroalga, Oedogonium occur

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under nitrogen limiting conditions, where internal nitrogen pools maintain growth for at least one week [15,16,41]. The ability of macroalgae to utilize their internal nitrogen reserves for growth and then rapidly replenish these reserves when external nitrogen becomes available suggests that maintaining nitrogen in excess to growth requirements may be unnecessary and could lead to reduced productivities and an increased proliferation of fouling organisms [12, 24]. Furthermore, it will also result in the unnecessary release of nitrogen in the discharge water from the algal cultures [15,39,54]. For freshwater macroalgae, it is unclear whether short term increases in productivity during acute nitrogen limitation [15,16,41] can be maintained when cultures are exposed to repeated cycles of nitrogendepletion and repletion over long periods, or whether chronic exposure to low nitrogen conditions imposes a physiological cost, such as a decline in photosynthetic capacity, resulting in lower cumulative productivity relative to cultures maintained under stable nitrogen conditions. The nitrogen content of the macroalgal biomass and the way nitrogen is supplied to cultures, either through discrete pulses or as a continuous supply, will also have a major influence on the biochemical profile of the biomass and ultimately determine how this biomass can be used. This is particularly important for macroalgae which have applications as an ingredient in animal feeds, as the quantity and quality of protein in the biomass is closely aligned to its internal nitrogen content and, in general, an increase in the internal nitrogen content results in a corresponding increase in the concentration of the amino acids which make up proteins [4,5,16]. Oedogonium is an attractive source of protein for animal agriculture as it has a high proportional composition of essential amino acids [16,40], those amino acids that need to be supplied to animals through their diet [9,10,55]. Of these essential amino acids, methionine and lysine are the most important as these are generally the firstlimiting amino acids for non-ruminant animals when fed plant-based diets of legumes and cereals. The amino acid content of Oedogonium is closely linked to its internal nitrogen content with the concentration of all amino acids (the sum of which is the protein content) declining when cultures are nitrogen-depleted [16]. While we expect the inverse of this relationship to also occur, i.e. the quantity of amino acids increases with increasing internal nitrogen content, there is limited understanding of the rate at which the concentrations of methionine, lysine and other essential amino acids recover following periods of nitrogen-depletion and repletion. This understanding will deliver a management strategy to manipulate the protein content of the algae even if the cultivation was conducted under nitrogen-limited conditions. Therefore, the overarching aim of this study was to quantify how variation in the supply of nitrogen to cultures of Oedogonium affects biomass productivity, photosynthetic capacity and the rate of protein recovery. To do this we undertook two complementary studies. Firstly, we assessed the longer-term effects of the manipulation of nitrogen supply on the physiological condition and biomass productivity of Oedogonium. The supply of nitrogen to cultures was manipulated over a 12-week period through the pulse addition of nitrogen such that cultures underwent repeated cycles of nitrogen supply in one, two or three-weekly additions. This enabled the determination of the chronic effects of nitrogen supply on biomass productivity, internal nitrogen content and photosynthetic capacity. Secondly, we determined the time taken for the internal nitrogen and amino acid contents of Oedogonium to recover following periods of nitrogen-depletion of increasing duration. These experiments enabled the development of a management strategy for the supply of nitrogen for the large scale production of Oedogonium biomass for its targeted use as a high protein feed ingredient. 2. Methods 2.1. Study species Oedogonium is a genus of unbranched filamentous green algae made up of small cylindrical cells. The genus has a worldwide distribution and

is a common component of natural ecosystems where it is found either attached to the substrate or as free floating mats. In intensive culture conditions Oedogonium is a robust and competitively dominant genera that has been identified as a key target group for the bioremediation of freshwater waste streams [17,28,29,43]. The biochemical composition of Oedogonium males it suitable for use as a feed ingredient for animal agriculture [15,16] or for bioenergy applications [40,41]. Stock cultures of Oedogonium sp. TSV2 (GenBank accession number: KF606977) as described in Lawton et al. [28], and hereafter referred to as Oedogonium, were sourced from stock cultures maintained at the Marine & Aquaculture Research Facilities Unit (MARFU), at James Cook University (JCU), Townsville (Latitude: 19.33°S; Longitude 146.76°E). This study was conducted over a 14-week period between 1st May and 14th August 2014 and during this period the water temperature and photosynthetically active radiation (PAR) was measured daily. The daily water temperature of the tanks followed the ambient air temperature and ranged between 15.9 and 26.8 °C. The PAR was measured at the surface of cultures using a flat panel Li-190SA Quantum sensor connected to a Li-1400 data logger (Li-Cor, Lincoln, NE, USA) and the mean daily PAR was 27.2 (± 0.7) mol photons m− 2, (range: 4.8– 36.6 mol m−2) with daily peaks ranging between 354 and 1870 μmol photons m− 2 s− 1. These environmental conditions were consistent within an experiment and all replicates experienced the same ambient environmental conditions. No attempt was made to manipulate these conditions as they are reflective of the environment that algae will be exposed to when it is cultured at large scales. 2.2. Experiment 1: manipulation of nitrogen supply To determine how biomass productivity, internal nitrogen content and the photosynthetic capacity of Oedogonium cultures are affected by the supply of nitrogen, a 12-week growth trial was undertaken in which nitrogen was supplied under three different treatments cycles of every week, every second week or every third week. This enabled the quanitification of both the immediate and longer term (chronic) effects that nitrogen supply has on the growth and photosynthetic capacity of Oedogonium. Oedogonium was cultured using nine cylindrical tanks (Duraplas AP1000; 1000 L capacity, 1.19 m2 surface area) filled to a depth of 75 cm with dechlorinated tapwater, with a volume of ~ 850 L. In each of these nine tanks 250 g of fresh biomass was added to give three replicate cultures per nitrogen supply treatment. To acclimatize the biomass to the experimental tank, and to provide baseline data on the photosynthetic efficiency of Oedogonium, each of the nine tanks were supplied with growth nutrients (Manutec MAF; nitrogen 13.4%, phosphorous 1.4%) during the first week at a rate of 0.1 g·L−1 (85 g) added on day one. Pulse amplitude modulated (PAM) fluorometry was used to determine the potential quantum yield (Fv/Fm) of photosystem II (PSII) as a direct measurement of light stress and the photosynthetic capacity of each replicate. A high Fv/Fm ratio means that a large proportion of the incoming light energy is captured and used for photosynthesis. Fv/Fm for each treatment was measured in dark-adapted samples, using a portable PAM fluorometer (Mini-PAM, Walz, Effeltrich, Germany). Five replicate samples from each replicate culture were placed in the fluorometer leaf-clip holder for darkadaptation (10 min) before a saturation pulse (approximately 4000 μmol photons m−2 s−1 for 0.4 s) was applied to determine Fv/ Fm. Measurements were taken at 12 pm on the day prior to harvest each week. To determine the biomass productivity of Oedogonium in g·dw·m−2·d−1, each culture was harvested weekly by draining the tank through a nylon bag. Harvested biomass was centrifuged (1000 rpm) to remove excess water and weighed to the nearest 0.1 g. Each culture was refilled with dechlorinated tapwater and restocked with fresh biomass at 0.3 g·L−1 (250 g) and, depending on the treatment cycle, nitrogen was added to these tanks through the addition of 85 g (0.1 g·L− 1) of MAF nutrients. Algal productivity was calculated

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using Eq. (1), where Bf and Bi are the final and initial biomass, FW:DW is the fresh weight (FW) to dry weight (DW) ratio, SA is the area of our culture tanks and t is the number of days in culture. For each culture the FW:DW was determined by drying a sample of centrifuged algae from each replicate at 60 °C for 48 h. The FW:DW ranged between 3.9 and 5.1. The internal nitrogen content of this biomass (2 g from each dried sample) was quantified using an elemental analyser (OEA Laboratory Ltd., Callington, UK) and is presented as percentage DW.  P¼


  B f −Bi =t: FW : DW  SA

ð1Þ

2.3. Statistical analysis The effect of nitrogen supply on the biomass productivity, photosynthetic capacity (Fv/Fm) and internal nitrogen content of the Oedogonium cultures was analysed using three, separate one-factor permutational analysis of variance (PERMANOVA). PERMANOVA is a nonparametric technique that partitions the total sums of squares to test for differences between groups [36]. It estimates a distance-based pseudo-F statistic and subsequently determines the P values based on permutational procedures. All PERMANOVA tests presented here are based on 10,000 permutations, using the unrestricted permutation of raw data and type III sums of squares using Primer 6 and the PERMANOVA+ add on [3]. As we were primarily interested in identifying the longer term effects of nitrogen supply, the data used in these analyses was the 12-week average for each of the three replicate tanks, which accommodated the differences in the length of nitrogen cycles between the three nitrogen treatments. The exception to this was the internal nitrogen content of the biomass in which the biomass from each of the three replicates was pooled each week to assess the range of values under nitrogen depletion and repletion. Instead we used the length of the nitrogen cycle as our level of replication for the nitrogen content analysis. This grouping does create an unbalanced design with n = 12 cycles for the weekly nitrogen treatment, n = 6 cycles for the treatment with nitrogen supplied every second week and n = 4 cycles for the treatment where nitrogen was supplied every third week. We note that one way PERMANOVA is robust to unbalanced designs and do not confound the analysis [3]. If there was a significant difference, pairwise a posteriori comparisons were made among the significant groups using the Bray–Curtis similarity measure (a = 0.05). 2.4. Experiment 2: recovery of internal nitrogen and amino acid content following nitrogen-depletion The second experiment assessed the rate of recovery of the internal nitrogen content of Oedogonium following nitrogen-depletion and whether there was any effect on the recovery of individual amino acids. To do this Oedogonium was cultured without nitrogen supplied for six time periods (96, 144, 192, 240, 288 and 336 h), after which each was supplied with nitrogen in excess (0.1 g·L−1 of MAF) and cultured for a further 7 days. This experiment was undertaken using the same sized tanks (1000 L) and again with three replicate cultures for each time treatment. Oedogonium was stocked in these tanks using the same density (0.3 g·L−1) as the first experiment. After each period of nitrogen-depletion these tanks were harvested and 250 g of this nitrogen-depleted biomass was restocked and MAF nutrients were provided at a rate of 0.1 g·L−1. To quantify the nitrogen content of the recovering biomass a 40 g FW sample of the biomass was taken from each tank 8 h after nitrogen addition and then in 24 h increments to give a time series of 0, 8, 24, 48, 72, 96, 120 and 144 h post supply of nitrogen. This sample was dried at 60 °C for 48 h. The biomass from each of the three replicate tanks was then combined in equal proportions for each time point and milled to a fine powder. One gramme of this pooled dry sample was used to

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determine the internal nitrogen content (OEA Labs) and 2 g was used for amino acid analysis (Australian Proteome Analysis Facility, Macquarie University, Sydney). Amino acids were analysed after liquid hydrolysis in 6 M HCl for 24 h at 110 °C using a Waters ACQUITY UPLC using procedures based on the Waters AccQTag amino acid methodology [11,14]. All samples were analysed for aspartic acid, asparagine, glutamic acid, glutamine, serine, histidine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline. As asparagine is hydrolysed to aspartic acid and glutamine to glutamic acid during analysis, the sum of these amino acids were reported as asparagine/aspartic acid or glutamine/ glutamic acid. Cysteine, tryptophan and taurine were not analysed as these require different analytical methods and represent only a very small fraction (b 2%) of the total amino acids present in macroalgae [4, 5]. The concentration of non-amino acid nitrogen is expressed in g 100 g−1 DW and was calculated using Eq. (2), where AAi is the concentration of the ith AA per 100 g DW biomass and Ni is the molecular % of nitrogen of the ith amino acid. Non AA N ¼ Total N−

XAAi  Ni  : 100

ð2Þ

3. Results 3.1. Experiment 1: manipulation of nitrogen supply 3.1.1. Biomass productivity The total productivity over the 12-week period differed significantly between the three nitrogen supply treatments (PERMANOVA F2,6 = 127.11, P = 0.004). The biomass productivity was highest in the treatment which received a weekly supply of nitrogen and lowest in the treatment that received nitrogen every third week. The weekly nitrogen treatment had a mean (± SE) productivity over the 12-week period of 10.56 (± 0.72) g DW·m− 2·day− 1 (Fig. 1a), which was 7.3% higher than when nitrogen was supplied every second week (9.84 ± 1.66 g DW·m− 2·day− 1) and 30.6% higher than when nitrogen was supplied every third week (8.09 ± 1.91 g DW·m− 2·day− 1). Biomass productivity was relatively stable in the weekly nitrogen treatment with productivity ranging between 8.88 (± 0.08) and 11.77 (± 0.73) g DW·m− 2·day− 1. In contrast the rate of biomass production was considerably more variable when nitrogen was supplied less frequently, with productivity ranging between 6.46 (± 0.17) and 13.09 (± 0.84) g DW·m− 2 day − 1 when nitrogen was supplied every second week and between 4.21 (± 0.43) and 13.14 (± 1.04) g DW·m− 2·day− 1 when nitrogen was supplied every third week. In these two treatments the lowest productivities consistently occurred during the weeks in which nitrogen was supplied, while the highest productivities occurred in the week following this for both treatments (Fig. 2a). The tanks that received nitrogen every third week had the most pronounced variation in growth, with the rate of biomass production decreasing to between 4.21 and 4.54 g·m− 2 day− 1 (Fig. 2a) during the second week of nitrogen depletion (Fig. 2a). Importantly this did not result in mortality in any of the replicate cultures. 3.1.2. Photosynthetic capacity The mean photosynthetic efficiency of cultures over the 12-week period differed significantly between the three nitrogen supply treatments (PERMANOVA F2,6 = 137.8, P = 0.004). The Fv/Fm ratio was highest in the weekly nitrogen treatment, averaging 0.71 (±0.02), compared to 0.60 (±0.03) and 0.52 (±0.07) when nitrogen was supplied every second and third week, respectively (Fig. 1b). The photosynthetic capacity was relatively stable in the weekly nitrogen treatment, with the Fv/Fm ratio ranging between 0.68 (± 0.03) and 0.78 (± 0.02) over the 12week period. In contrast, as the period without nitrogen increased the

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content was highest in the weekly nitrogen treatment at 5.6 (±0.2) % compared to 3.9 (±1.1) % when nitrogen was supplied every second week and 3.2 (±1.2) % when nitrogen was supplied every third week (Fig. 1c). The internal nitrogen content was relatively stable in the weekly nitrogen supply treatment but varied considerably between weeks in the other two treatments (Fig. 2c). In the weekly nitrogen treatment the internal nitrogen content of the biomass ranged between 4.9 and 6.2%, whereas the internal nitrogen content ranged between 1.7 and 5.8% when nitrogen was supplied every second week and between 1.2 and 6.0% when nitrogen was supplied every third week (Fig. 2c). During the first week of nitrogen-depletion the internal nitrogen content of the biomass in both the second and third weekly supply treatments declined by between one half and two thirds to range between 1.6–2.4% (Fig. 2c). During the week that nitrogen was supplied to the second weekly and third weekly nutrient treatments, the internal nitrogen content of the biomass rapidly recovered and returned to a level that was equivalent to the weekly nitrogen treatment (Fig. 2c). 3.2. Experiment 2: recovery of internal nitrogen and amino acid content following nitrogen-depletion

Fig. 1. The effect of changing the supply of nitrogen on the a) the biomass productivity and b) the photosynthetic capacity (Fv/Fm) and c) internal nitrogen content of Oedogonium cultures over a 12 week period. These cultures were maintained under three different nitrogen environments where nitrogen was supplied every week, every second week or every third week. The data is the mean (±SE) of replicate (n = 3) cultures over the 12week period. Bars that share a common letter do not differ significantly, PERMANOVA, Pairwise comparison P b 0.05.

Fv/Fm ratio decreased to a minima of 0.51 (±0.02) and 0.32 (±0.03) when nitrogen was supplied every second and third week, respectively (Fig. 2b). However, the photosynthetic capacity of cultures rapidly increased following the re-supply of nitrogen and during these weeks the Fv/Fm ratio ranged between 0.58 (±0.03) and 0.68 (±0.02) and between 0.59 (±0.04) and 0.62 (±0.03) in the treatments which had nitrogen added every second and third week, respectively (Fig. 2b). 3.1.3. Internal nitrogen content The mean internal nitrogen content of the biomass over the 12-week period differed significantly between the three nitrogen supply treatments (PERMANOVA F2,20 = 191.7, P b 0.001). The internal nitrogen

3.2.1. Internal nitrogen decline and recovery The internal nitrogen content of the biomass decreased over a 14day period from a mean (± SE) of 5.09 (± 0.15) to 0.96 (± 0.08) % (Fig. 2). The most rapid decrease occurred in the first 96 h with the internal nitrogen content decreasing to 2.11 (± 0.05) %, a decline of more than 50%. The rate and magnitude of the decline in nitrogen content slowed markedly beyond this point, with the mean nitrogen content decreasing by less than a factor of 0.2 for each subsequent 48 h increment beyond 96 h (Fig. 3). After these cultures were resupplied with nitrogen their internal nitrogen content rapidly increased and within 8 h the internal nitrogen content had increased by a factor of between 1.5 and 2.1-times. Within 24 h the internal nitrogen content had increased by a factor of between 2.2 and 3.9-times with the fastest rate of increase occurring in the cultures which had the longest period of nitrogen-depletion. After 24 h all treatments had recovered between 70 and 90% of their pre-depleted nitrogen (Fig. 3). At this point the 96-, 144- and 192-h depletion treatments had an internal nitrogen content that ranged between 4.58 (±0.04) and 4.68 (±0.53) %, while the 240-, 288- and 336-h depletion treatments had a nitrogen content of 4.15 (±0.09), 4.26 (±0.03) and 3.59 (±0.05) %, respectively (Fig. 3). The internal nitrogen content continued to increase in all treatments beyond 24 h but the rate of increase slowed considerably after 48 h and by 72 h the internal nitrogen content of the biomass in all treatments had reached an asymptote in concentration that ranged between 5.2–5.8% (Fig. 2). The final concentrations were slightly higher than the initial nitrogen content (5.09%) of the original biomass. 3.2.2. Amino acid recovery After a period of nitrogen-depletion that ranged between 96- and 336-h the biomass had a total amino acid content that ranged between 10.23 and 5.13 g · 100 g.−1 with the lowest concentrations of amino acids in biomass that had the longest period of nitrogen-depletion (Fig. 4). After nitrogen was resupplied to these cultures the total amino acid content recovered rapidly (Table 1). Within 8 h the concentration of the total amino acids had increased by between 50 and 90% and within 24 h the total amino acid content in all treatments had increased 2–3 fold and ranged between 15.22 and 20.71 g · 100 g− 1 (Fig. 4a). The rate of increase in the total amino acid content was fastest for those treatments which had the longest period of nitrogen-depletion and, consequently, the lowest initial concentrations of amino acids. However, after 48 h the rate of increase in the total amino acid content for all nitrogen-depletion treatments slowed considerably and reached an asymptote between 24.12 and 26.51 g · 100 g−1 (Fig. 4a). The increase in the concentration of the essential amino acids was delayed slightly with only a minor increase (b15%) occurring during

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Fig. 2. Week to week effects of changing the supply of nitrogen on a) the biomass productivity, b) internal nitrogen content and c) the photosynthetic capacity (Fv/Fm) of Oedogonium cultures. These cultures were maintained under three different nitrogen environments where nitrogen was supplied every week, every second week or every third week. On the bars N denotes the weeks in which nitrogen was added to each of the three treatments. The data for productivity and Fv/Fm is the mean (±SE) of the three replicate tanks, while the internal nitrogen content is a pooled sample from each (n = 1) of the three replicate cultures for each week.

the first 8 h. However, between 8 and 24 h after nitrogen was resupplied the concentration of the essential amino acids had increased by a factor of between 1.8 and 2.5 with the concentration of the essential amino acids ranging between 5.65 and 8.21 g · 100 g−1 after 24 h. The concentration of the essential amino acids continued to increase at a relatively fast rate and approached an asymptote in concentration 72 h after nitrogen was resupplied to these cultures (Fig. 4b). The increase in the

concentration of the essential amino acids after 72 h was minimal and beyond 96 h all treatments converged upon a concentration that ranged between 10.26 and 11.37 g · 100 g−1 (Fig. 4b, Table 1). In general the initial recovery of total amino acids and essential amino acids in the treatments that were nitrogen-depleted for 240-, 288- and 336-h lagged behind the recovery of the treatments that were nitrogendepleted for shorter periods (96-, 144- and 192-h) by approximately

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Fig. 3. The rate of decline and recovery of the internal nitrogen content of Oedogonium cultures when depleted of nitrogen for an increasing period of time: 96, 144, 192, 240, 288 and 336 h (black line), before being resupplied with nitrogen (grey lines). The recovery of the internal nitrogen content was assessed at 8 h after nitrogen was added and then every 24 h for the next 7 days. Data is the mean (±SE) of three replicate cultures.

24 h. However, the former treatments had a faster rate of recovery and reached the same endpoint in the concentrations of the amino acids (Fig. 4a, b). The initial increase in the concentration of the total amino acids during the first 8 h after nitrogen was resupplied is almost entirely due to an increase in the concentration of glutamine/glutamic acid (Fig. 4c).

The largest increase of glutamine/glutamic acid occurred in the treatment that was nitrogen-depleted for 336-h (14 days) which increased from 0.68 to 4.57 g·100 g−1. In general, after an initial 8 h peak, the concentration of glutamine/glutamic acid in all treatments decreased to a stable concentration that ranged between 2.72 and 3.73 g · 100 g− 1 (Fig. 4c). The exception to this was the 288-h nitrogen-depletion

Fig. 4. Changes in a) total amino acid, b) essential amino acid, c) glutamine/glutamic acid, d) lysine, e) methionine, and f) non-amino acid nitrogen concentrations in Oedogonium biomass after being resupplied with nitrogen following a nitrogen-depletion period (DP) of increasing duration: 96, 144, 192, 240, 288 or 340 h. The recovery of amino acids was assessed 8 h after nitrogen was added and then every 24 h for the next 7 days. Data is a pooled sample of the three replicate cultures (n = 1) at each time point.

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Table 1 Concentration of the internal nitrogen content, non-amino acid nitrogen and amino acids in the Oedogonium biomass (% DW). The data shown is the range (minimum and maximum) across the six nitrogen-depletion treatments (96, 144, 192, 240, 288 or 340 h before being resupplied with nitrogen). Data in parenthesis is the range in the proportion of the total amino acids that each amino acid accounts for at each time point. % DW

Concentration after nitrogen-depletion

72 h after nitrogen resupplied

144 h after nitrogen resupplied

Nitrogen Non AA nitrogen Total AA Total EAA Alanine Aspartic acid Arginine Glutamic acid Glycine Histidinea Isoleucinea Leucinea Lysinea Methioninea Phenylalaninea Prolinea Serine Threoninea Tyrosine Valinea

0.96–2.11 0.21–0.59 5.13–10.29 2.23–4.55 0.35–0.71 (6.82–7.06) 0.55–1.08 (10.43–11.87) 0.28–0.59 (5.39–5.94) 0.68–1.35 (13.13–13.62) 0.29–0.56 (5.44–5.65) 0.09–0.23 (1.75–2.17) 0.24–0.49 (4.42–4.69) 0.47–0.99 (9.07–9.64) 0.39–0.73 (6.98–7.60) 0.1–0.19 (1.78–1.95) 0.3–0.66 (5.81–6.38) 0.27–0.65 (5.14–5.47) 0.29–0.53 (5.1–5.65) 0.3–0.57 (5.5–5.85) 0.19–0.38 (3.56–3.97) 0.34–0.69 (6.41–6.76)

5.14–5.48 1.66–2.01 22.16–25.2 9.83–11.01 1.47–1.74 (5.82–6.52) 2.45–2.99 (9.72–11.87) 1.32–1.51 (5.24–6.00) 2.84–3.72 (10.9–13.28) 1.19–1.35 (4.72–5.34) 0.47–0.54 (1.87–2.14) 1.08–1.23 (4.27–4.86) 2.2–2.44 (8.73–9.68) 1.60–1.8 (6.69–7.30) 0.40–0.46 (1.59–1.8) 1.37–1.57 (5.44–6.13) 1.13–1.26 (4.48–4.99) 1.1–1.21 (4.36–4.78) 1.22–1.32 (4.76–5.22) 0.79–0.97 (3.13–3.86) 1.51–1.69 (5.95–6.72)

5.42–5.88 1.69–2.02 25.06–26.51 10.99–11.35 1.64–1.74 (6.52–6.96) 2.93–3.09 (12.55–13.65) 1.47–1.51 (5.66–5.96) 3.14–3.45 (12.55–13.65) 1.33–1.4 (5.2–5.44) 0.52–0.55 (2.00–2.13) 1.19–1.25 (4.71—4.81) 2.47–2.56 (9.39–9.77) 1.75–1.84 (6.87–7.21) 0.37–0.46 (1.46–1.85) 1.59–1.64 (5.89–6.44) 1.30–1.33 (4.93–5.32) 1.28–1.33 (4.92–5.25) 1.36–1.41 (5.26–5.44) 0.94–1.08 (3.67–4.21) 1.65–1.73 (6.48–6.58)

a

Essential amino acids.

treatment. In this treatment the concentration of glutamine/glutamic acid continued to increase over the first 24 h and peaked at 7.12 g · 100 g− 1, which was higher than treatments that were nitrogen-depleted for both shorter and longer timeframes. Following this peak the concentration of glutamine/glutamic acid in the 288-h treatment decreased steadily and by 72 h had converged upon the other five treatments. The concentration of glutamine/glutamic acid in all treatments at the end of the experimental period ranged between 3.14 and 3.36 g · 100 g−1 (Fig. 3c). The concentration of the essential amino acids lysine and methionine exhibited a similar increase when cultures were resupplied with nitrogen. Neither of these amino acids increased during the first 8 h following the resupply of nitrogen. However, between 8 and 72 h the concentration of both exhibited a steady linear increase (Fig. 4d, e). This trend was mirrored by all other quantified amino acids (Table 1, Supp. A). The concentration of lysine increased between 2 and 4 times, up to 1.8 g · 100 g−1 after 72 h (Fig. 4d). Methionine also increased between 2 and 4 times, up to 0.46 g · 100 g−1 after 72 h (Fig. 4e). For both of these amino acids the largest changes in magnitude and the highest rate of increase occurred in the treatments which had the longest period of nitrogen-depletion (Fig. 4d, e). The concentration of lysine continued to increase beyond 72 h albeit at a much slower rate and all of the nitrogen-depletion treatments converged upon an asymptote concentration of up to 1.85 g·100 g−1 (Fig. 4d, Table 1). In contrast the concentration of methionine between 72 and 144 h was more variable with up to a 15% change in concentration occurring within a treatment between sampling times. During this time period the methionine content of all treatments oscillated within a band that ranged between 0.37 and 0.48 g · 100 g−1 (Fig. 4e). In addition to the nitrogen converted into amino acids there is also a proportion of nitrogen that is not incorporated into amino acids. The concentration of non-amino acid nitrogen in the Oedogonium biomass exhibited a steady linear increase for the first 24 h after nitrogen was added. Over this time period the non-amino acid nitrogen content increased by a factor between 3 and 6, from an initial concentration that ranged between 0.21–0.59 g · 100 g−1 to a concentration after 24 h of between 1.32–1.81 g · 100 g− 1. The lowest concentration of nonamino acid nitrogen was in the treatments that had the longest period of nitrogen-depletion. The rate of increase slowed beyond 24 h and approached an asymptotic concentration after 48 h that ranged between 1.5–2 g · 100 g− 1, equivalent to between 30 and 35% of the total nitrogen content (Fig. 4f, Table 1).

4. Discussion This study has demonstrated that intensive cultures of Oedogonium are highly robust to changes in the external supply of nitrogen and that this biomass can survive for at least 2 weeks without external nitrogen. Further there was no significant reduction in biomass productivities when nitrogen was supplied every second week. Interestingly in these treatments the rate of biomass production was enhanced by up to 20% during the first week of nitrogen depletion. However, extending the period of nitrogen-depletion to two weeks (nitrogen supplied every third week treatment), had a negative impact on cultures with reduced biomass productivity and photosynthetic capacity. During periods of nitrogen-depletion both the concentration of internal nitrogen and the concentration of amino acids steadily decreased at a rate inverse to that of biomass productivity. This result conforms to previous observations of marine macroalgae (seaweed) where free amino acids (FAA) form one of the main storage pools of nitrogen and that this nitrogen can be metabolized to synthesize new structural and metabolic proteins to maintain their growth [8,38,48]. Prolonged periods of nitrogendepletion significantly impaired the photosynthetic capacity of the biomass, supporting that these cultures were under physiological stress relative to cultures that were maintained in an environment of more regular nitrogen supply. Despite this, an important result of this is study is that, regardless of the length of the nitrogen-depletion period, the photosynthetic efficiency, internal nitrogen content and the concentrations of the essential amino acids were all rapidly replenished when nitrogen was resupplied. Furthermore, there were no chronic detrimental effects on any of these cultures following repeated cycles of nitrogendepletion and repletion. The increase in biomass productivity that was consistently observed during the first week of nitrogen-depletion is a result of unbalanced growth whereby the processes of nutrient acquisition are uncoupled from those of carbon fixation [7]. During such periods the energetic costs of nutrient acquisition and assimilation are absent, increasing the carbon available for biomass growth. This has the effect of increasing growth rates during the initial stages of nitrogen-depletion and may also explain similar observations in other studies of Oedogonium [16, 41]. However, if the period between nitrogen supply is extended to three weeks, then the biomass will dilute its internal nitrogen with carbohydrates below a critical point upon which growth becomes limited by the internal nitrogen content [5]. In this study, the biomass productivity and photosynthetic capacity of Oedogonium was unchanged

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during the first week of nitrogen-depletion (week two of the second weekly and third weekly supply treatments), but decreased significantly during the second week of nitrogen-depletion (week three of the third weekly supply treatment). This suggests that the critical nitrogen content for Oedogonium growing at approximately 12 g·DW m−2·d−1 is between 1.34 and 2.01% DW. This is similar to the critical nitrogen content of 1.2% determined for the green seaweed Ulva ohnoi growing at similar rates [5], and demonstrates that Oedogonium can efficiently utilize stored nitrogen to maintain growth rates during periods of restricted nitrogen supply. The critical nitrogen content in this study is specific to the environmental conditions of light, temperature and carbon supply, however, it can be used as a general guide for assessing the extent to which Oedogonium can be depleted of nitrogen before a decrease in the rate of biomass production occurs. During periods when external nitrogen is available Oedogonium replenishes its internal nitrogen concentration, but this comes at an energetic cost with a short-term decrease in the rate of biomass production. In plants under normal conditions, the metabolic cost of the uptake and assimilation of nitrogen represents between 25 and 52% of their total energy budget [22,42,53] and this cost is expected to be higher following periods of nitrogen-depletion. In this study the photosynthetic capacity of the cultures that were exposed to nitrogen-depletion for one week were similar to the cultures which had nitrogen supplied weekly and, as such, the reduction in biomass productivity of the second week of nitrogen-depletion cycles is expected to be entirely due to the increased metabolic cost associated with the uptake and assimilation of this nitrogen. However, following a second week of nitrogendepletion in the third weekly supply treatment, we then saw that the photosynthetic capacity of cultures decreased substantially. This result suggests that the reduced growth rate in this treatment is a function of both the metabolic costs of nitrogen assimilation and of nitrogen limitation due to the internal nitrogen content falling below the critical nitrogen content (decreasing proteins, chlorophyll etc.), resulting in a lower overall mean productivity for the 12-week period. In contrast, the Oedogonium that was only exposed to one week of nitrogendepletion had a total productivity (9.84 ± 0.78 g DW·m− 2·day−1) that was not significantly different to the weekly nitrogen treatment, suggesting that growth was never nitrogen-limited. This highlights the robust nature of Oedogonium where it can accommodate variation in the nutrient concentration of incoming wastewater without incurring a significant loss in productivity.

Nitrogen-depleted Oedogonium initially caused a large increase in glutamine/glutamic acid within the first 8 h. This spike in glutamine/ glutamic acid also occurs in seaweeds [5,25,48] and represents the first stages of inorganic nitrogen assimilation by plants and algae via the glutamate dehydrogenase (GDH) and glutamine synthetase/glutamine: 2-oxoglutarate aminotransferase (GS/GOGAT) pathways (Fig. 5) [19]. As inorganic nitrogen must first be reduced to ammonium before it can be assimilated and converted into organic nitrogen, it is expected that inorganic nitrogen or non-amino acid nitrogen would spike rapidly after nitrogen repletion and before glutamine/glutamic acid. We did not expect to observe this spike with an initial sampling period of 8 h, however, we did observe that glutamine/glutamic acid was converted into the other amino acids over the subsequent 64 h and by 72 h the total amino acid content of this biomass had been fully restored (Fig. 5). Furthermore, the concentrations of the essential amino acids, including methionine and lysine, had recovered to their maximum concentrations at this same time point. As methionine and lysine are often the first limiting amino acids when animals are fed plant-based diets [10,37], the recovery of these amino acids may be considered as indicators for the complete nutritional recovery of the biomass. As methionine is the start codon for the synthesis of protein [19], its recovery in particular can also be used as a more specific indicator of protein recovery in the biomass [5]. This ability to rapidly restore the quantity and quality of amino acids following periods of nitrogen-depletion is a natural adaptation to the fluctuating nitrogen availability found in natural ecosystems [31] and, as demonstrated in this study, can be used to facilitate the rapid recovery of the nutritional value of nitrogen-depleted Oedogonium biomass. These results support previous amino acid repletion studies performed for seaweeds [25,38,48] and for the first time demonstrate that freshwater macroalgae restore essential amino acids over short (b3 days) time periods when cultivated under intensive conditions and growing at high biomass productivities. The nitrogen content of the algal biomass and, more specifically, the quantity and quality of amino acids are the major factors in determining the range of bioproduct applications for the biomass. Biomass that has a low concentration of nitrogen is preferred for biofuel applications as the presence of nitrogenous compounds both interferes with catalysts during conventional refining and forms harmful oxide emissions when the fuel is combusted [41]. Conversely, biomass that has a high concentration of nitrogen, and therefore a high concentration of amino acids, is preferable for use as animal feed supplements [16,54]. The results of

+ − + Fig. 5. The process of uptake and assimilation of inorganic N (NO− 3 and NH4 ) into intracellular organic N involves (A) the uptake and reduction of NO3 (nitrate-nitrogen) into NH4 (ammonia-nitrogen), (B) the assimilation of inorganic N into organic N through the formation of free amino acids (FAA) by the synthesis of glutamic acid and glutamine via the glutamate dehydrogenase (GDH) and glutamine synthetase/glutamine: 2-oxoglutarate aminotransferase (GS/GOGAT) pathway [19], (C) the synthesis of other amino acids from glutamic acid and glutamine via a multitude of pathways, and (D) the synthesis of proteins and other nitrogenous organic macromolecules.

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this study demonstrate that the cultivation of Oedogonium is highly adaptable and robust to periods where external nitrogen is unavailable, growing for two weeks without the addition of nitrogen and that the pulse addition of nitrogen is a viable culture technique that can produce Oedogonium biomass that is suitable for energy or agricultural applications. Importantly our results demonstrate that Oedogonium biomass can rapidly oscillate between a nitrogen content of 2 and 5.5% without any significant loss in the rate of biomass production over the longer term. It is unlikely that our results for macroalgae are transferable to microalgae, as while the biomass productivities are often the same, the higher stocking density and lower growth rates of macroalgae contrast with the typically much higher (2–3 times) growth rates of microalgae which results in a much faster dilution rate of nitrogen [1, 30]. The concentration of the amino acids measured in this study are the highest recorded for freshwater macroalgae with a total amino acid content that ranged between 25.5 and 26.5 g · 100 g−1. Likewise, the concentration of the essential amino acids methionine (0.40– 0.46 g · 100 g− 1) and lysine (1.75–1.85 g · 100 g− 1) were 10–20% higher than previous measures which ranged between 0.29–0.43 and 1.23–1.52 g · 100 g−1 for methionine and lysine, respectively [16,40]. These higher amino acid concentrations were correlated with the higher nitrogen content of the biomass which ranged between 5.4– 5.8% compared to 4.16 and 4.8% in the previous studies of Oedogonium [16,40]. This higher internal nitrogen content is most likely due to the relatively higher supply of nitrogen and slower growth rates which act together to concentrate nitrogen and amino acids in macroalgal biomass [5,6]. These results confirm that the quantity of essential amino acids in Oedogonium biomass is comparable to terrestrial sources [10, 15] and that these amino acids are not altered by using nutrient pulsing. The ability to effectively culture Oedogonium using nutrient pulses has significant implications for its commercial production at multihectare scales. One of the drivers for commercializing freshwater macroalgae is that the intensive cultivation in nutrient-rich wastewater can provide a water treatment service in addition to generating revenue as a source of protein. The industrial ecology of siting algal facilities adjacent to wastewater producers is particularly applicable to intensive animal agriculture where large quantities of nutrient-rich waste are produced [34,44,46]. In circumstances where the nutrient stream is concentrated, it may not be practical or economical to provide a continuous supply of water/nitrogen. Instead, pulse additions can provide the same nutrient flux (i.e. concentration by flow) while maintaining biomass quality and with limited impact on biomass productivity. As nutrient extraction from the wastewater is a function of the total amount of biomass produced and its nitrogen content, there will be no reduction in wastewater treatment outcomes using pulsed feeding techniques. Rather, it may even be higher as no nitrogen will be lost through discharge water. However, what will differ is the rate of nutrient uptake. When nitrogen is always available the rate of nitrogen uptake is constant and ranges between 1.09–1.22 g·m− 2·day− 1 for Oedogonium [15,16]. In contrast, when nitrogen is added as a pulse to nitrogen-depleted biomass the rate of uptake is rapid with the biomass becoming nitrogenreplete within 48 h. In conclusion, Oedogonium biomass can be successfully cultivated over the longer term using the pulse addition of nitrogen with minimal impacts on the rate of biomass production and no long term effects on the concentration of essential amino acids. Negative impacts on productivity were only pronounced when the internal nitrogen content of the biomass was depleted to below the critical nitrogen content of between 1.3–2%, which for Oedogonium occurred during a second week of nitrogen-depletion. Importantly, when this nitrogen-depleted biomass was re-supplied with nitrogen it rapidly replenished its internal nitrogen content which returned to pre-depleted levels within 24–48 h. Moreover this increase in internal nitrogen content was rapidly translated into amino acids, including the essential amino acids methionine and lysine which returned to pre-depleted levels within 72 h. This result

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has implications for the logistics of nutrient delivery and highlights the ability to manipulate the composition of Oedogonium for use in energy and feed applications. Importantly, for animal feed applications the quality and quantity of the amino acids in the biomass can be rapidly upgraded by providing a pulse of nitrogen 72 h prior to harvest. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2015.10.010. Acknowledgements This research is part of the MBD Energy Research and Development Programme for Biological Carbon Capture and Storage. This project is supported by the Advanced Manufacturing Cooperative Research Centre (AMCRC), funded through the Australian Government's Cooperative Research Centre Scheme, and the Australian Renewable Energy Agency (ARENA) (002369). We thank Jonathan Moorhead, Giovanni del Frari, Thomas Mannering for assisting with the experiments. References [1] A.L. Ahmad, N.H.M. Yasin, C.J.C. Derek, J.K. Lim, Microalgae as a sustainable energy source for biodiesel production: a review, Renew. Sust. Energ. Rev. 15 (2011) 584–593. [2] H. Aiking, Future protein supply, Trends Food Sci. Technol. 22 (2011) 112–120. [3] M.J. Anderson, R.N. Gorley, K.R. Clarcke, PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods, PRIMER-E, Plymouth, UK, 2008. [4] A.R. Angell, I. Pirozzi, R. de Nys, N.A. Paul, Feeding preferences and the nutritional value of tropical algae for the abalone Haliotis asinina, PLoS One 7 (2012), e38857. [5] A.R. Angell, L. Mata, R. Nys, N.A. Paul, Variation in amino acid content and its relationship to nitrogen content and growth rate in Ulva ohnoi (Chlorophyta), J. Phycol. 50 (2014) 216–226. [6] A.R. Angell, L. Mata, R. de Nys, N.A. Paul, Indirect and direct effects of salinity on the quantity and quality of total amino acids in Ulva ohnoi (Chlorophyta), J. Phycol. (2015) 536–545. [7] I. Berman-Frank, Z. Dubinsky, Balanced growth in aquatic plants: myth or reality? Bioscience 49 (1999) 29–37. [8] K.T. Bird, C. Habig, T. DeBusk, Nitrogen allocation and storage patterns in Gracilaria tikvahiae (Rhodophyta), J. Phycol. 18 (1982) 344–348. [9] S. Boisen, T. Hvelplund, M.R. Weisbjerg, Ideal amino acid profiles as a basis for feed protein evaluation, Livest. Prod. Sci. 64 (2000) 239–251. [10] M.J. Boland, A.N. Rae, J.M. Vereijken, M.P. Meuwissen, A.R. Fischer, M.A. van Boekel, S.M. Rutherfurd, H. Gruppen, P.J. Moughan, W.H. Hendriks, The future supply of animal-derived protein for human consumption, Trends Food Sci. Technol. 29 (2013) 62–73. [11] L. Bosch, A. Alegría, R. Farré, Application of the 6-aminoquinolyl-Nhydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods, J. Chromatogr. B 831 (2006) 176–183. [12] T. Capo, J. Jaramillo, A. Boyd, B. Lapointe, J. Serafy, Sustained high yields of Gracilaria (Rhodophyta) grown in intensive large-scale culture, J. Appl. Phycol. 11 (1999) 143–147. [13] A.F. Clarens, E.P. Resurreccion, M.A. White, L.M. Colosi, Environmental life cycle comparison of algae to other bioenergy feedstocks, Environ. Sci. Technol. 44 (2010) 1813–1819. [14] S.A. Cohen, Amino acids analysis using precolumn derivatisation with 6aminoquinolyl-N-hydroxysuccinimidyl carbamate, in: C. Cooper, N. Packer, K. Williams (Eds.), Methods Mol Biol, Humana Press, Totowa, NJ 2001, pp. 39–47. [15] A.J. Cole, R. de Nys, N.A. Paul, Removing constraints on the biomass production of freshwater macroalgae by manipulating water exchange to manage nutrient flux, PLoS One 9 (2014), e101284. [16] A.J. Cole, R. de Nys, N.A. Paul, Biorecovery of nutrient waste as protein in freshwater macroalgae, Algal Res. 7 (2015) 58–65. [17] A.J. Cole, L. Mata, N.A. Paul, R. de Nys, Using CO2 to enhance carbon capture and biomass applications of freshwater macroalgae, Glob. Change Biol. Bioenergy 6 (2013) 637–645. [18] J.S. Craigie, Seaweed extract stimuli in plant science and agriculture, J. Appl. Phycol. 23 (2011) 371–393. [19] R.H. Garrett, C.M. Grisham, Biochemistry, 5th edition Brooks/Cole, Cengage Learning, 2013. [20] K.W. Gellenbeck, Utilization of algal materials for nutraceutical and cosmeceutical applications—what do manufacturers need to know? J. Appl. Phycol. 24 (2012) 309–313. [21] H.C.J. Godfray, J.R. Beddington, I.R. Crute, L. Haddad, D. Lawrence, J.F. Muir, J. Pretty, S. Robinson, S.M. Thomas, C. Toulmin, Food security: the challenge of feeding 9 billion people, Science 327 (2010) 812–818. [22] V.P. Gutschick, Evolved strategies in nitrogen acquisition by plants, Am. Nat. 118 (1981) 607–637. [23] J.T. Hafting, A.T. Critchley, M.L. Cornish, S.A. Hubley, A.F. Archibald, On-land cultivation of functional seaweed products for human usage, J. Appl. Phycol. 24 (2012) 385–392.

486

A.J. Cole et al. / Algal Research 12 (2015) 477–486

[24] M.D. Hanisak, The use of Gracilaria tikvahiae (Gracilariales, Rhodophyta) as a model system to understand the nitrogen nutrition of cultured seaweeds, Hydrobiologia 204 (1990) 79–87. [25] A.B. Jones, W.C. Dennison, G.R. Stewart, Macroalgal responses to nitrogen source and availability: amino acid metabolic profiling as a bioindicator using Gracilaria edulis(Rhodophyta), J. Phycol. 32 (1996) 757–766. [26] E. Kebede-Westhead, C. Pizarro, W.W. Mulbry, Treatment of swine manure effluent using freshwater algae: production, nutrient recovery, and elemental composition of algal biomass at four effluent loading rates, J. Appl. Phycol. 18 (2006) 41–46. [27] B.E. Lapointe, Strategies for pulsed nutrient supply to Gracilaria cultures in the Florida Keys: interactions between concentration and frequency of nutrient pulses, J. Exp. Mar. Biol. Ecol. 93 (1985) 211–222. [28] R.J. Lawton, R. de Nys, N.A. Paul, Selecting reliable and robust freshwater macroalgae for biomass applications, PLoS One 8 (2013), e64168. [29] R.J. Lawton, R. de Nys, S. Skinner, N.A. Paul, Isolation and identification of Oedogonium species and strains for biomass applications, PLoS One 9 (2014), e90223. [30] Y. Liang, N. Sarkany, Y. Cui, Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions, Biotechnol. Lett. 31 (2009) 1043–1049. [31] C.S. Lobban, P.J. Harrison, Seaweed Ecology and Physiology, Cambridge University Press, USA, 1997. [32] J.N. Lott, J. Kolasa, G.D. Batten, L.C. Campbell, The critical role of phosphorus in world production of cereal grains and legume seeds, Food Secur. 3 (2011) 451–462. [33] L. Machado, R.D. Kinley, M. Magnusson, R. de Nys, N.W. Tomkins, The potential of macroalgae for beef production systems in Northern Australia, J. Appl. Phycol. 27 (2015) 2001–2005. [34] J. Martinez, P. Dabert, S. Barrington, C. Burton, Livestock waste treatment systems for environmental quality, food safety, and sustainability, Bioresour. Technol. 100 (2009) 5527–5536. [35] L. Mata, M. Magnusson, N.A. Paul, R. de Nys, The intensive land-based production of the green seaweeds Derbesia tenuissima and Ulva ohnoi: biomass and bioproducts, J. Appl. Phycol. (2015) 1–11. [36] B.H. McArdle, M.J. Anderson, Fitting multivariate models to community data: a comment on distance-based redundancy analysis, Ecology 82 (2001) 290–297. [37] P. McDonald, R.A. Edwards, J.F.D. Greenhalgh, C.A. Morgan, Animal nutrition, Pearson Education Limited, Essex, 2002. [38] K.J. McGlathery, M.F. Pedersen, J. Borum, Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum (Chlorophyta), J. Phycol. 32 (1996) 393–401. [39] W. Mulbry, S. Kondrad, C. Pizarro, E. Kebede-Westhead, Treatment of dairy manure effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers, Bioresour. Technol. 99 (2008) 8137–8142.

[40] N. Neveux, M. Magnusson, T. Maschmeyer, R. de Nys, N.A. Paul, Comparing the potential production and value of high-energy liquid fuels and protein from marine and freshwater macroalgae, Glob. Change Biol. Bioenergy (2014). [41] N. Neveux, A. Yuen, C. Jazrawi, M. Magnusson, B. Haynes, A. Masters, A. Montoya, N. Paul, T. Maschmeyer, R. de Nys, Biocrude yield and productivity from the hydrothermal liquefaction of marine and freshwater green macroalgae, Bioresour. Technol. 155 (2014) 334–341. [42] F.W.T. Penning de Vries, The cost of maintenance processes in plant cells, Ann. Bot. 39 (1975) 77–92. [43] D.A. Roberts, R. de Nys, N.A. Paul, The effect of CO2 on algal growth in industrial waste water for bioenergy and bioremediation applications, PLoS One 8 (2013), e81631. [44] C. Rotz, Management to reduce nitrogen losses in animal production, J. Anim. Sci. 82 (2004) 119–137. [45] J. Ryther, N. Corwin, T. DeBusk, L. Williams, Nitrogen uptake and storage by the red alga Gracilaria tikvahiae (McLachlan, 1979), Aquaculture 26 (1981) 107–115. [46] J. Sims, L. Bergström, B. Bowman, O. Oenema, Nutrient management for intensive animal agriculture: policies and practices for sustainability, Soil Use Manag. 21 (2005) 141–151. [47] V. Smil, Phosphoros in the environment: natural flows and human interferences, Annu. Rev. Energy Environ. 25 (2000) 53–88. [48] M.W. Taylor, N.G. Barr, C.M. Grant, T.A.V. Rees, Changes in amino acid composition of Ulva intestinalis (Chlorophyceae) following addition of ammonium or nitrate, Phycologia 45 (2006) 270–276. [49] P.K. Thornton, Livestock production: recent trends, future prospects, Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 365 (2010) 2853–2867. [50] D. Tilman, K.G. Cassman, P.A. Matson, R. Naylor, S. Polasky, Agricultural sustainability and intensive production practices, Nature 418 (2002) 671–677. [51] D. Tilman, J. Fargione, B. Wolff, C. D'Antonio, A. Dobson, R. Howarth, D. Schindler, W.H. Schlesinger, D. Simberloff, D. Swackhamer, Forecasting agriculturally driven global environmental change, Science 292 (2001) 281–284. [52] A. van Huis, Potential of insects as food and feed in assuring food security, Annu. Rev. Entomol. 58 (2013) 563–583. [53] P. White, Introduction, definition and classification of nutrients, in: P. Marschner (Ed.), Mineral Nutrition of Higher Plants, Academic Press, Boston, MA 2012, pp. 7–44. [54] A.C. Wilkie, W.W. Mulbry, Recovery of dairy manure nutrients by benthic freshwater algae, Bioresour. Technol. 84 (2002) 81–91. [55] G. Wu, Amino acids: metabolism, functions, and nutrition, Amino Acids 37 (2009) 1–17.