Culture of Spirulina platensis in human urine for biomass production and O₂ evolution

Culture of Spirulina platensis in human urine for biomass production and O 2 evolution first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:wuzc@zju.edu.cn †E-mail:wuzc@zju.edu.cn wuzc@zju.edu.cn Department of Environmental Engineering, Zhejiang University, Hangzhou 310027, China Department of Environmental Engineering, Zhejiang University, Hangzhou 310027, China Attempts were made to culture Spirulina platensis in human urine directly to achieve biomass production and O 2 evolution, for potential application to nutrient regeneration and air revitalization in life support system. The culture results showed that Spirulina platensis grows successfully in diluted human urine, and yields maximal biomass at urine dilution ratios of 140~240. Accumulation of lipid and decreasing of protein occurred due to N deficiency. O 2 release rate of Spirulina platensis in diluted human urine was higher than that in Zarrouk medium. Spirulina platensis Human urine Biomass production O 2 evolution Life support system INTRODUCTION 1 For long distance manned space missions, biological life support systems (BLSS) have to be introduced into the life support system to achieve regeneration of nutrient matter (Volker and Frank, 2001 2001 ). Recently, many kinds of subsystems within BLSS have been proposed and experimented on solely or jointly, which include mainly higher plant, aquatic organisms (Volker and Frank, 2001 2001 ), microorganisms and microalgae (Gros et al., 2003 2003 ). Microalgae were incorporated in many BLSS because of their high growth rate, simple cultivation and relatively easy collection (Gitelson and Rodicheva, 1993 1993 ). Among the microalgae, Spirulina platensis is the most commonly employed because of its rich nutrition and potential for substitution for conventional food (Ciferri and Tiboni, 1985 1985 ). In Micro Ecological Life Support System Alternative (MELSSA), a first attempt was made to use treated waste, mainly including feces and human urine, to culture Spirulina platensis (Gros et al., 2003 2003 ). In the system, three steps biological pre-treatment of the waste was applied, so that the complete system became somewhat complicated. Some organic matters in feces, such as fiber, are inefficient for bio-treatment. Human urine contains minimal organic matters, but much inorganic matter, such as N, P and K (Larsen et al., 2001 2001 ), and so, is ideal culture medium for the photoautotrophic Spirulina platensis . We reported here our attempts at culturing Spirulina platensis in human urine solely and directly to achieve nutrient regeneration and air revitalization. MATERIALS AND METHODS 1 Spirulina platensis 439 was obtained from the Freshwater Algae Bank of the Chinese Academy of Sciences (CAS). Culture medium used in this study includes Zarrouk medium (ZM) (Gòdia et al., 2002 2002 ), synthetic human urine (SHU) (Gordon, 1982 1982 ) and real human urine (RHU). ZM (1000 ml) is composed of 18.0 g NaHCO 3 , 2.5 g NaNO 3 , 0.5 g K 2 HPO 4 , 1.0 g K 2 SO 4 , 1.0 g NaCl, 0.04 g CaCl 2 , 0.08 g Na 2 EDTA, 0.2 g MgSO 4 ·7H 2 O, 0.01 g FeSO 4 ·7H 2 O and 1.0 ml trace elements (TE). TE (g/L): H 3 BO 3 2.86; (NH 4 ) 6 Mo 7 O 24 0.02; MnCl 2 ·4H 2 O 1.8; Cu 2 SO 4 0.08; ZnSO 4 ·7H 2 O 0.22. The culture medium pH was adjusted to 8.2 by 1 mol/L NaOH solution. SHU (1000 ml) was composed of 0.5 g CaCl 2 ·2H 2 O, 4.12 g K 2 HPO 4 , 0.47 g MgCl 2 ·H 2 O, 0.29 g KCl, 4.83 g NaCl, 1.55 g NH 4 Cl, 2.37 g Na 2 SO 4 , 13.34 g urea, 1.0 g creatinine and 0.65 g sodium citrate (pH 6.8). A bubble column photobioreactor with total culture volume of 1200 ml was designed and used to culture Spirulina platensis . Culture condition was set as follows light intensity 444.4 w/m 2 , aeration rate 1.8 L/min, illuminating time 14.0 h/d and temperature 30 °C. Spirulina platensis in ZM and log phase was collected and inoculated into different culture medium to start the culture process, during which, deionized water was added daily to compensate for water loss by evaporation. Water, protein, lipid, total chlorophyll, carotenoid, dissolved oxygen (DO) and ash were analyzed following standard methods (APHA et al., 1995 1995 ; Sullivan and Carpenter, 1993 1993 ). Metallic elements, including K, Na, Ca, Mg and Fe were determined using ICP-AES (Inductively coupled plasma-atomic emission spectrometry). DO increasing rate in ZM and 180-diluted RHU were measured in closed photobioreactor. Before staring the experiments, O 2 was removed by purging with N 2 for 15 min at rate of 6 L/min. CO 2 (100 ml) was then injected into the bottom of the reactor. Recirculation (2 L/min) was started to avoid biomass sedimentation and improve mass transfer. Inoculated biomass concentration was about 0.6 g/L, light intensity 444.4 w/m 2 and temperature 30 °C. Linear growth rate ( v ) (Ogbonna et al., 1995 1995 ) was introduced to evaluate the growth rate of Spirulina platensis , and can be calculated according to the equation: v= ( DW t − DW i )/ t , where v is the linear growth rate, t is the culture time, DW i and DW t is the dry weight at the beginning and end of the linear growth phase respectively; each growth curve was calculated after logistic curve fitting. The maximum value of the curve is then defined as the maximum productivity ( P max ), used to evaluate the maximal biomass concentration. RESULTS AND DISCUSSION 1 Results of batch culture of Spirulina platensis in 180-diluted RHU, 180-diluted SHU and commonly used ZM are shown in Fig. 1 1 . Spirulina platensis grew successfully and yielded similar culture results in 180-diluted RHU ( v =0.266 g/(L·d), P max =2.32 g/L) and SHU ( v =0.236 g/(L·d), P max =2.40 g/L), and their growth curves showed no statistical different (95% confidence level). Moreover, their main compositions were on the same level (Table 1 1 ). Composition (100 g dry wt) 180-diluted RHU 180-diluted SHU ZM Water (g) 4.98 5.23 4.23 Protein (g) 32.40 34.78 67.20 Lipid (g) 20.43 17.58 11.93 Total chlorophyll (mg) 1429.19 1408.56 1399.68 Carotenoid (mg) 227.99 269.49 337.76 Ash (g) 4.42 4.50 6.50 K (mg) 229.31 245.66 564.52 Na (mg) 457.18 483.49 723.44 Ca (mg) 332.25 336.19 65.84 Mg (mg) 726.35 648.87 1337.68 Fe (mg) 5.79 19.45 37.08 Except for 3 d additional lag phase, Spirulina platensis in these three culture media showed similar culture characteristics, growing exponentially in log phase, and stabilized in stationary phase. P max in 180-diluted RHU and SHU was lower than that in ZM (3.74 g/L), which might have resulted from the nutrient shortage. The composition of Spirulina platensis in 180-diluted RHU was rich in lipid (20.43%), but deficient in protein (32.40%), in contrast with that in ZM which were 11.93% and 67.20% correspondingly. 180-diluted RHU (C:N:P molar ratio: 20.04:21.6:1) was apparently deficient in C but rich in N, in comparison with ZM (C:N:P molar ratio: 74.66:10.25:1). During batch-culture, CO 2 from air can be used as C source. Due to evaporation loss of NH 4 -N (Carvalho et al., 2004 2004 ), N shortage appeared (N:P molar ratio: 2.8:1) when the culture process entered into log phase, and subsequently synthesis of amino acids and protein was inhibited. Fig. 2 2 showed the effect of dilution ratio on the growth of Spirulina platensis . P max gets its peak within the range of about 140~240. Low P max in low dilution ratio may result from inhibition effects of high NH 4 -N concentration (Carvalho et al., 2004 2004 ). And low P max in high dilution ratio may due to scarcity of nutrient matters. DO increasing in ZM and in 180-diluted RHU in the closed photobioreactor are shown in Fig. 3 3 . DO rose sharply at first, but finally stabilized near their maximal values (ZM: 7.01 mg/L; 180-diluted RHU: 6.80 mg/L). Michaelis-Menten equation (Ritchie and Prvan, 1996 1996 ) could describe the effect of dissolved CO 2 concentration ( S CO 2 ) on DO increasing rate ( v DO ): $$v_{{\text{DO}}} {\text{ = }}{{v_{{\text{DOmax}}} S_{{\text{CO}}_{\text{2}} } } \mathord{\left/{\vphantom {{v_{{\text{DOmax}}} S_{{\text{CO}}_{\text{2}} } } {(K_{S_{{\text{CO}}_{\text{2}} } } {\text{ + }}S_{{\text{CO}}_{\text{2}} } )}}} \right.\kern-\nulldelimiterspace} {(K_{S_{{\text{CO}}_{\text{2}} } } {\text{ + }}S_{{\text{CO}}_{\text{2}} } )}}$$ where K S CO 2 is Michaelis-Menten constant. At the beginning, S CO 2 was high enough, and the equation is simplified to v DO = v DOmax , which means a zero-order increase in DO as was the case in both culture media within the initial 60 min. DO increasing can be described as: d DO / d t = K DO , where K DO is the reaction constant and t is the reaction time. Then, K DO in ZM and 180-diluted RHU was calculated to get the value of 49.30 and 62.75 μg/(L·min) respectively, which show a higher O 2 release rate in 180-diluted RHU. CONCLUSION 1 Spirulina platensis was successfully grown in diluted RHU to achieve biomass production and O 2 evolution. Thus, it is possible to use Spirulina platensis to regenerate nutrient matter, and at the same time, to assimilate CO 2 and release O 2 within the life support system. References APHA (American Public Health Association) AWWA (American Water Works Association) WEF (Water pollution Control Federation) Standard Methods for Water and Wastewater Examination , 1995 . : Washington DC . 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