Bioresource Technology 193 (2015) 219–226
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Stabilisation of microalgae: Iodine mobilisation under aerobic and anaerobic conditions Wei Han a, William Clarke a,b, Steven Pratt a,⇑ a b
School of Chemical Engineering, University of Queensland, Queensland, Australia School of Civil Engineering, University of Queensland, Queensland, Australia
h i g h l i g h t s First investigation into iodine mobilisation during algae stabilisation. Useful amounts of iodine are retained during aerobic and anaerobic stabilisation. I mobilisation is correlated with C emission, indicating I present as organoiodine. N compared to I mobilisation indicates N and I are housed separately in algae.
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 9 June 2015 Accepted 13 June 2015 Available online 19 June 2015 Keywords: Iodine biofortification Algal fertiliser Iodine mobilisation Aerobic digestion Anaerobic digestion
a b s t r a c t Mobilisation of iodine during microalgae stabilisation was investigated, with the view of assessing the potential of stabilised microalgae as an iodine-rich fertiliser. An iodine-rich waste microalgae (0.35 ± 0.05 mg I g1 VSadded) was stabilised under aerobic and anaerobic conditions. Iodine mobilisation was linearly correlated with carbon emission, indicating iodine was in the form of organoiodine. Comparison between iodine and nitrogen mobilisation relative to carbon emission indicated that these elements were, at least in part, housed separately within the cells. After stabilisation, there were 0.22 ± 0.05 and 0.19 ± 0.01 mg g1 VSadded iodine remaining in the solid in the aerobic and anaerobic processed material respectively, meaning 38 ± 5.0% (aerobic) and 50 ± 8.6% (anaerobic) of the iodine were mobilised, and consequently lost from the material. The iodine content of the stabilised material is comparable to the iodine content of some seaweed fertilisers, and potentially satisfies an efficient I-fertilisation dose. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Iodine deficiency is a worldwide health concern. In 1980, about 60% of the world’s population were iodine deficient (Zimmermann et al., 2008); iodized salt has since been supplemented into daily diets but still 30% of the world’s population suffered iodine deficiency in 2007 (de Benoist et al., 2008). Additional strategies for iodine supplementation have been considered (Smolen´ et al., 2014b). For example, increasing the iodine content of eatable plants was thought to be a safer and more efficient option than industrial iodized salt, as 75–85% of the iodine in human body comes from vegetable food under natural conditions (Weng et al., 2008). Increasing iodine in plants – iodine biofortification – is facilitated by using iodine-rich fertilisers.
⇑ Corresponding author. E-mail address: [email protected] (S. Pratt). http://dx.doi.org/10.1016/j.biortech.2015.06.063 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Using iodine rich fertilisers can lead to some fruit and vegetables having an iodine content of several mg kg1 (dry weight), e.g. carrot: 1 mg kg1, tomato: 6 mg kg1, soybean: 3.5 mg kg1 (Dai et al., 2004; Weng et al., 2014); leaf vegetables tend to have relatively higher iodine content, e.g. spinach: 65 mg kg1, celery: 50 mg kg1, Chinese cabbage: 80 mg kg1 (Nkemka and Murto, 2010; Singh and Ajay, 2011). Researchers have shown a linear relationship between iodine uptake rate and exogenous iodine (I, IO 3) level (0–10 mg L1) for cabbage and celery (Hong et al., 2009). Foliar fertilisation with KIO3 at 1 mg I L1 can result in lettuce leaves having an iodine content of 800 mg kg1 (Smolen´ et al., 2014a), which is comparable to some high iodine content seaweeds (Kupper et al., 2008). Algae have long been recognised as an important iodine accumulator in natural environments (Kupper et al., 2008). With the increasing interest in iodine biofortification, algae of high iodine content have been considered as a fertiliser. For example, ground dry kelp (0.2–0.3% iodine) was found to be an effective fertiliser
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for increasing iodine content in soil and improving iodine uptake by plants (Weng et al., 2008). The study showed kelp fertilisation of 40 mg kg1 (iodine to soil) could provide a residual iodine content of 25 mg kg1 in soil (free iodine) after 90 days plantation. Meanwhile, during the plantation, the iodine content of eatable plants was elevated to significant levels, e.g. cucumber: 6 mg kg1, aubergine: 10 mg kg1, radish: 8 mg kg1. Weng et al’s. (2008) study also found kelp as an iodine fertiliser can slow down iodine release in soil compared with potassium iodide (KI). Every year there are large amounts of waste algae generated from aquatic systems in many places around the world (Han et al., 2014). Algal fertiliser for iodine biofortification could represent a new opportunity for utilisation of waste algae with high iodine content. However, before applying to land, organic waste must be stabilised to avoid adverse environmental consequence (Polprasert, 1996). Stability refers to the rate or degree of organic matter decomposition, and can be expressed as a function of microbiological activity (Wu et al., 2000). It has been concluded that stabilisation increases the agricultural value of the material as a consequence of organic matter humification, reduces pathogens, and destroys some toxic compounds (Sanchez-Monedero et al., 2004). But there are currently no reports concerning iodine mobilisation during algae stabilisation. In this study, iodine-rich waste microalgae harvested from a groundwater holding pond was stabilised under both aerobic and anaerobic conditions. The organic carbon degradation and nutrient and iodine mobilisation were monitored. This is the first work considering iodine mobilisation during organic waste stabilisation. The outcomes are significant in the context of utilising the final products as fertiliser for iodine biofortification. 2. Methods In this work, ‘waste algae’ refers to algae harvested from a laboratory algal growth system (the laboratory growth system represents a groundwater holding pond, with iodine rich groundwater used as the growth medium). The waste algae was stabilised in (i) an aerobic environment, using activated sludge from a wastewater treatment plant as the inoculum for stabilisation, and (ii) an anaerobic environment, using digester sludge from a wastewater treatment plant as the inoculum for stabilisation. 2.1. Waste algae and inoculum for stabilisation Waste microalgae was collected from a lab scale production system that used iodine-rich groundwater (2.0 mg iodide L1) as the water source. The lab scale algal production system consisted of a 5 L large glass beaker illuminated with florescent light of 80 lmol m2 s1 at 25 °C using the groundwater as medium. After 4 months cultivation, the microalgae biofilm in the beaker was harvested and drained on a 500 lm pore size mesh plate. Then the mesh plate with algae was placed in a 4 °C fridge overnight for further drainage. To decompose the waste microalgae, wastewater treatment sludge was used as inoculum. For aerobic digestion, activated sludge was obtained from the aerobic zone at Luggage Point wastewater treatment plant (LPWWTP), Brisbane, Queensland, Australia. For anaerobic digestion, digester sludge was obtained from the same treatment plant. 2.2. Waste algae stabilisation Carbon emission was used as the measure of stabilisation. Stability refers to the rate or degree of organic matter decomposition, and can be expressed as a function of microbiological activity
(Wu et al., 2000), which in this case was determined by CO2 and CH4 production. The experimental set-ups for waste algae stabilisation are shown in Fig. 1. For aerobic digestion, 30 g wet algae, 15 g inoculum and 115 g distilled water were mixed in a 240 ml glass reactor. A magnetic stirrer was used for mixing. As shown in Fig. 1, input air flowed through two 240 ml glass bottles, the first containing saturated NaOH solution (200 ml), the second distilled water (200 ml), to ensure a CO2 free and water saturated air feed. After each reactor, there was another 240 ml glass bottle containing 100 ml NaOH solution (2 mol L1) for trapping the CO2 from exhaust gas. The four bottles were connected by tygon tubing in series for one way ventilation by compressed air. The aerobic stabilisation trial was repeated 4 times. The negative control, which was carried out in duplicate, contained additional water in place of the algae. Detailed set-up conditions are shown in Table 1. For anaerobic digestion, 30 g wet algae, 70 g inoculum and 60 g distilled water were mixed in 240 ml glass bottles, which were then sealed with butyl rubber stoppers. Before being sealed, the headspace was flushed with nitrogen gas. The anaerobic stabilisation trial was also repeated 4 times. The group of negative controls (triplicate) contained additional water in place of the algae. An active control experiment (duplicate) was also performed to verify the activity of the inoculum, by using 1.1 g cellulose as the substrate instead of algae.
2.3. Sampling and sample analysis The characteristics of the materials before and after stabilisation were analysed. During the stabilisation, a 1.5 ml liquid sample was taken from each reactor every one or two weeks. Total I, N, P and pH were measured in the liquid samples. To monitor waste microalgae biomass degradation during aerobic stabilisation, the NaOH solutions in the exhaust line were analysed for CO2 each time liquid was sampled from the reactors. After each sample, the exhaust gas traps were emptied and filled with fresh NaOH solution. For anaerobic stabilisation, a 6 ml gas sample from the headspace of each reactor was taken and measured while sampling the liquid. Carbon and nitrogen content of the algae was analysed by a FLASH 2000 CHNS/O Analyser. Compositions of gas samples were analysed by a Shimadzu GC-2014. For analysing CO2 produced by aerobic stabilisation, 1 ml NaOH solution from the exhaust gas traps was added to a sealed vacuum tube (12.5 ml capacity). Then each vacuum tube was filled with 4 ml of 4 M H2SO4 solution to ensure release of CO2 under acidic conditions. The CO2 content of the headspace of the vacuum tube was measured by GC. In the aquatic environment, the species of elemental iodine vary as a function of pH and redox conditions (Gottardi, 2001). Also, soluble organic iodine compounds may be present. So to quantify the total iodine mobilised into the liquid phase, tetramethylammonium hydroxide (TMAH) was used to pre-treat the samples to ensure all the iodine species were converted to inorganic iodide (Fecher et al., 1998). 0.1 ml of filtered liquid sample was mixed with 0.1 ml 25% TMAH solution. The mixture was then heated in a water bath at 90 °C for three hours. After cooling, the liquid samples were then diluted by distilled water to 1 ml and then analysed by a Dionex ICS-3000 Ion Chromatography system with an AS-18 column. For the detection of iodine content in algae, wet biofilm samples were rinsed with distilled water and dried at 110 °C overnight. 0.2 g dry algal biofilm solid was digested following a procedure reported by Ródenas de la Rocha et al. (2009). After digestion, the iodine content in the liquid was measured by the ion chromatography method. Nitrogen and phosphorus content were monitored by Flow Injection Analysis (FIA).
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Fig. 1. Experimental set-up for waste algae stabilisation.
Table 1 Set-up conditions of waste algae stabilisation. Process
Material
AG
NC
AC
Water (%)
VS (%)
Temp.(°C)
Air supply (ml min1)
Aerobic digestion
Algae Sludge Distilled water
30 g 15 g 115 g
0 15 g 145 g
0 0 0
91.9 98.9 100
7.8 0.9 0
25
35
Anaerobic digestion
Algae Sludge Distilled water Cellulose
30 g 70 g 60 g 0
0 70 g 90 g 0
0 70 g 90 g 1.1 g
91.8 97.8 100 0
7.8 2.1 0 100
37
0
AG: algae group. NC: negative control. AC: active control.
Table 2 Characteristics of initial materials used in the waste microalgae stabilisation experiments (±SD). Process
Group
Phase
Mass (g)
I content (ppm)
C (ppm)
N* (ppm)
P (ppm)
Total COD L + S (mg)
Aerobic
Algae
L S L S
0.02 ± 0.00 a: 350 ± 50 s: 14.3 0.00 ± 0.00 s: 14.3
0 a: 374000 s: 419000 0 s: 419000
6.89 ± 1.2 a: 65000 s: 84000 8.28 ± 1.49 s: 84000
9.09 ± 0.96 a: 6000 s: 12000 4.23 ± 1.24 s: 12000
2646 ± 243
NC
158.42 a: 2.44 ± 0.24 s: 0.17 ± 0.02 159.84 s: 0.17 ± 0.02
Algae
L S
NC
L S
157.12 a: 2.42 ± 0.24 s: 1.55 ± 0.07 158.53 s: 1.55 ± 0.07
0.43 ± 0.03 a: 350 ± 50 s: 51.7 0.30 ± 0.01 s: 51.7
0 a: 374000 s: 358000 0 s: 358000
304 ± 8.50 a: 65000 s: 57000 329 ± 7.10 s: 57000
29.6 ± 3.71 a: 6000 s: 20000 26.8 ± 0.75 s: 20000
Anaerobic
187.0 ± 0.94 4127 ± 275
1686 ± 33
NC: negative control; L: liquid phase; S: solid phase, all values in solid phase are on a dry weight basis. * Liquid phase nitrogen was the sum of nitrate-N and ammonium-N; a: values in algae; s: values in inoculum sludge.
2.4. Kinetic model Considering algae material consists of rapidly and slowly degradable components (Bai et al., 2014), a two-substrate model was applied in this study, with production of gas i (i = CH4, CO2) being described as follows:
Y i ðtÞ ¼ Y i;rapid ð1 ekrapid t Þ þ Y i;slow ð1 ekslow t Þ
ð1Þ
where Y i ðtÞ is the cumulative yield of a gas i at time t (ml g1 VSadded) (VSadded, volatile solid added to system); Y i;rapid is the maximum potential gas yield from the rapidly degradable substrate (ml g1 VSadded); krapid is hydrolysis rate constant for the degradation of rapidly degradable substrate (d1); Y i;slow is the maximum potential gas yield from the slowly degradable substrate (ml g1 VSadded); kslow is hydrolysis rate constant for the degradation of slowly degradable substrate (d1); and t is the time (d). The parameters were obtained from data fitting using the SigmaPlot 12.0 software.
The carbon emission from the aerobic and anaerobic stabilisations was determined from the sum of carbon dioxide and methane production (Aerobic: CO2-C; Anaerobic: CH4-C + CO2-C). i.e.,
X
EðtÞ ¼ M C
Y i ðtÞ
ð2Þ
i¼CH4 ;CO2
where EðtÞ is the cumulative carbon emission at time t (mg g1 VSadded); MC is the molar volumetric concentration of carbon (12/24.5 mg ml1, 25 °C). The carbon emission rate was used to determine the stability of organic wastes during a stabilisation process (Cabañas-Vargas et al., 2005; Kalamdhad et al., 2008). In this study, a model calculated value was used to indicate the carbon emission rate:
Erate ðtÞ ¼ EðtÞ0 where Erate ðtÞ is the (mg g1 VSadded d1).
ð3Þ carbon
emission
rate
at
time
t
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2.5. Mass balance calculation In a closed system, the elemental mass balance is:
Si C is ðxÞ þ Li C il ðxÞ þ Gi C ig ðxÞ ¼ Sf C fs ðxÞ þ Lf C fl ðxÞ þ Gf C fg ðxÞ
ð4Þ
where Si ; Li ; andGi are the initial mass of solid (dry basis), liquid, and gas phases (kg); Sf ; Lf ; and Gf are the final mass of solid (dry basis), liquid, and gas phases (kg); C is ðxÞ; C il ðxÞ; and C ig ðxÞ are the initial concentrations of the element in the solid (dry basis), liquid, and gas phases (mg kg1); and C fs ðxÞ; C fl ðxÞ; and C fg ðxÞ are the final concentrations in the solid (dry basis), liquid, and gas phases (mg kg1); In this study, mass balances were carried out in two steps. Firstly, a phosphorus mass balance was carried out to determine the actual initial dry solid mass Si in each reactor. P content in algae C is ðPÞ (dry basis) was measured as a highly repeatable value with very small standard deviations. Mass balances were then carried out for elemental C, I and N. The following parameters were assumed to be zero: C il ðCÞ, C ig ðCÞ, C ig ðIÞ, and C ig ðNÞ (CO2 and N2 in air were not considered). 3. Results and discussion 3.1. Characteristics of waste algae The harvested microalgae biofilm was mostly green and banded. Some of the biofilm that was harvested from the shade side of the cultivation beaker was brown. Under the microscope, the microalgae biofilm is characterised as a mat of green filamentous organisms with isolated cells of different morphologies mixed within the mat. The characteristics of the microalgae are shown in Table 2. The iodine content of the material was 350 ± 50 mg kg1. This value is much lower than that of kelp (2000–3000 mg kg1) (Weng et al., 2008), but still comparable to the iodine content in many reported seaweed species (Romaris-Hortas et al., 2011). Such an iodine content will potentially satisfy an efficient I-fertilisation dose, which has been reported to be 40 mg I per kg soil (Weng et al., 2008). Still, it is acknowledged that it would be important to study the in-field mineralisation of iodine in stabilised algae to confirm that the iodine would ultimately be available for plants. A feature of waste algae is that it is rich in nitrogen (Han et al., 2014). The nitrogen content (6.5%) of the waste microalgae in this study was higher than that of many organic wastes (Komilis et al., 2012). The content of phosphorous (0.6%) in waste microalgae was lower than the P content (1–2%) in some P-rich organic wastes and sludges (García-Romero et al., 2014; Rughoonundun et al., 2010; Wilkie and Mulbry, 2002), but higher than many reported waste algae materials (Bucholc et al., 2014; Ibrahim et al., 2014). These
Table 3 Kinetic parameters of biogas production using two-substrate model. Parameters
Fig. 2. Tracking carbon through the stabilisation processes. a: Carbon emission during aerobic and anaerobic stabilisation, (s) aerobic, (d) anaerobic; b: biogas production in anaerobic stabilisation, () CH4, (4) CO2; c: carbon mass balance, with liquid phase C determined as the difference between the initial C and the measured final solid and gas phase C, ( ) solid phase, (h) gas phase, (j) liquid phase (liquid phase carbon was obtained via calculation).
Y i;rapid (ml g1 VSadded) krapid (d1) Y i;slow (ml g1 VSadded) kslow (d1) Overall potential maximum biogas yield Y i;max (ml g1 VSadded) Overall potential maximum carbon emission Emax (mg g1 VSadded) Measured carbon emission after stabilisation (mg g1 VSadded) R2
Aerobic
Anaerobic
CO2
CH4
CO2
100.8 0.0183 111.4 0.0183 212.2
77.5 0.416 65.0 0.0318 142.5
37.1 0.0942 40.0 0.0102 77.1
103.9
69.8
37.8
87.6
69.6
31.2
0.983
0.997
0.999
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W. Han et al. / Bioresource Technology 193 (2015) 219–226 Table 4 Characteristics of final stabilisation products. Process
Group
Phase
Aerobic
Algae
L S L S
NC Anaerobic
Algae NC
L S L S
Mass (g) 158.6 1.20 ± 0.04* 159.9 0.05 ± 0.02 157.41 2.27 ± 0.12 158.70 1.03 ± 0.01
I content (ppm)
C (ppm)
N* (ppm)
1.37 ± 0.33 436.9 ± 92.4 0.01 ± 0.00 ND
ND 349800 ± 15700 ND 357700 ± 15600
273 ± 41.5 59300 ± 6100 70.3 ± 0.70 46000 ± 9900
45.9 ± 25.0 8900 ± 3200 20.3 ± 0.44 ND
1264 ± 126
3.05 ± 0.45 215.8 ± 17.2 0.45 ± 0.00 37.6 ± 1.1
ND 362600 ± 8600 ND 370800 ± 7800
992 ± 3.90 57100 ± 2000 596 ± 6.03 51400 ± 3900
90.2 ± 43.8 15300 ± 2100 84.6 ± 6.23 21100 ± 500
2102 ± 386
P (ppm)
Total COD L + S (mg)
83 ± 11
1340 ± 17
NC: negative control; ND: non detection. L: liquid phase; S: solid phase, all values in solid phase are on a dry weight basis. * Liquid phase nitrogen was the sum of nitrate-N and ammonium-N.
characteristics show the waste microalgae in this study to be potentially a useful fertiliser. 3.2. Stabilisation Algae was stabilised in both aerobic and anaerobic environments. Carbon emission, shown in Fig. 2a, was used as a measure of degradation. The kinetics differed but this was at least in part due to substantial differences in the substrate to inoculum ratios. It was assumed that stabilisation was ultimately first-order, which allowed for the determination of the extent of degradation and carbon emission rate. By the end of the trials (after 100 days), about 50% of the total CODadded of the algae was degraded (Tables 2 and 4). There was 21.9 ± 5.0% and 25.6 ± 1.9% (mean value ± SD) carbon emitted and the model calculated emission rates were 0.24 mg C g1 VSadded d1 (day100) and 0.10 mg C g1 VSadded d1 (day105) for the aerobic and anaerobic environments respectively, indicating that the material was ‘stable’ by the end of the trials (Cabañas-Vargas et al., 2005; Kalamdhad et al., 2008). Kinetic analysis was applied to help estimate the degradation potential of waste microalgae under the current stabilisation conditions. The calibrated parameters are shown in Table 3. The results show that the two-substrate model fits the measured values of carbon emission and biogas production (Fig. 2a and b). It seems there were no significant differences between rapidly and slowly degradable substrates in aerobic conditions, as the calibrated hydrolysis rate constants of both rapidly and slowly degradable substrates appeared to be the same value (0.0183 d1, Table 3). The rate constants (kCH4 ;rapid , kCH4 ;slow , kCO2 ;rapid ) for anaerobic stabilisation were higher than that for aerobic stabilisation (Table 3). Model simulation revealed Emax was 103.9 and 106.6 mg g1 VSadded in aerobic and anaerobic stabilisations respectively. The measured extent of carbon emission was 87.6 mg g1 VSadded (aerobic) after 100 days and 100.9 mg g1 VSadded (anaerobic) after 105 days. As such, most of the readily degradable substrates were degraded within 100 days. The methane production during anaerobic stabilisation was 140 ± 10 ml g1 VSadded or 130 ± 10 ml g1 CODadded (Fig. 2b). This yield was similar to some reported values for algae material that was stabilised anaerobically without pre-treatment (Allen et al., 2013; Nkemka and Murto, 2010). Carbon mass balance showed 172 ± 9.2 mg g1 VSadded (aerobic) and 182 ± 12 mg g1 VSadded (anaerobic) carbon remained in solid phase after degradation, which indicated 54 ± 7.3% (aerobic) and 52 ± 3.7% (anaerobic) of the total carbon was mobilised (Fig. 2c). 3.2.1. Mobilisation of iodine To examine the potential of using waste microalgae as an algae iodine fertiliser, it is important to understand the mobilisation of iodine during stabilisation. The degradation of the microalgae resulted in a total iodine level in the liquid of 0.56 ± 0.14
mg L1 g1 VSadded in the aerobic environment and 1.26 ± 0.19 mg L1 g1 VSadded in the anaerobic environment (Fig. 3a). The initial iodine content of the microalgae for both the aerobic and anaerobic stabilisations was similar (0.35 ± 0.05 mg g1 VSadded) (Fig. 3b). At the end of the stabilisations, 0.22 ± 0.05 mg g1 VSadded iodine remained in the solid phase in the aerobic environment while 0.19 ± 0.01 mg g1 VSadded iodine remained in the solid phase in the anaerobic environment (Fig. 3b). Iodine mobilisation in the aerobic environment (38 ± 5.0%) was less than in the anaerobic environment (50 ± 8.6%). Considering the degree of mobilised carbon was similar in both processes (around 50%, Fig. 2c), the results indicate iodine in waste microalgae is less readily mobilised during aerobic stabilisation. There was a strong linear correlation between the liquid phase iodine content and the emitted carbon in both environments (aerobic: R2 = 0.9901; anaerobic: R2 = 0.9914) (Fig. 3c). This indicated elemental iodine in waste microalgae was probably covalently bound to organic compounds rather than stored in the form of iodide. The latter had been reported in a few macroalgae species. From some macroalgae species, stored iodide can be released into the extracellular environment within a very short time (several hours) under specific environmental stimulations (normally oxidative stress) (Kupper et al., 2008; Nitschke et al., 2013). Organically bound iodine was reported in many living organisms, especially species in terrestrial water systems (Gribble, 2003; Li et al., 2013). Dehalogenation during the degradation of organic halocompounds can be an important process that causes release of halides (van Pée and Unversucht, 2003). A possible explanation for the lower level of iodine mobilisation in aerobic conditions is a difference in the mechanisms of dehalogenation under aerobic and anaerobic conditions. It has been shown that microbial catalysed dehalogenation under aerobic conditions is usually complicated and dependent on the level of NADH, while under anaerobic conditions an organic halocompound can serve as a terminal electron acceptor to form up a halo-respiration chain (van Pée and Unversucht, 2003). 3.2.2. Mobilisation of nutrients It is important to consider the mobilisation of nutrients if the stabilisation products are to be used as organic fertilisers. Liquid phase nitrogen was present as nitrate (NO 3 ) in aerobic conditions and as ammonium (NH+4) in anaerobic conditions (Fig. 4a and b). During stabilisation, decomposition of proteins results in release of ammonia. In the aerobic environment the mobilised ammonia is oxidised to NO 3 , which reduces pH (Hamoda and Ganczarczyk, 1980). The average pH in the aerobic environment was 6.8 at the beginning, with the pH eventually dropping to
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Stabilisation of microalgae: Iodine mobilisation under aerobic and anaerobic conditions Wei Han a, William Clarke a,b, Steven Pratt a,⇑ a b
School of Chemical Engineering, University of Queensland, Queensland, Australia School of Civil Engineering, University of Queensland, Queensland, Australia
h i g h l i g h t s First investigation into iodine mobilisation during algae stabilisation. Useful amounts of iodine are retained during aerobic and anaerobic stabilisation. I mobilisation is correlated with C emission, indicating I present as organoiodine. N compared to I mobilisation indicates N and I are housed separately in algae.
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 9 June 2015 Accepted 13 June 2015 Available online 19 June 2015 Keywords: Iodine biofortification Algal fertiliser Iodine mobilisation Aerobic digestion Anaerobic digestion
a b s t r a c t Mobilisation of iodine during microalgae stabilisation was investigated, with the view of assessing the potential of stabilised microalgae as an iodine-rich fertiliser. An iodine-rich waste microalgae (0.35 ± 0.05 mg I g1 VSadded) was stabilised under aerobic and anaerobic conditions. Iodine mobilisation was linearly correlated with carbon emission, indicating iodine was in the form of organoiodine. Comparison between iodine and nitrogen mobilisation relative to carbon emission indicated that these elements were, at least in part, housed separately within the cells. After stabilisation, there were 0.22 ± 0.05 and 0.19 ± 0.01 mg g1 VSadded iodine remaining in the solid in the aerobic and anaerobic processed material respectively, meaning 38 ± 5.0% (aerobic) and 50 ± 8.6% (anaerobic) of the iodine were mobilised, and consequently lost from the material. The iodine content of the stabilised material is comparable to the iodine content of some seaweed fertilisers, and potentially satisfies an efficient I-fertilisation dose. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Iodine deficiency is a worldwide health concern. In 1980, about 60% of the world’s population were iodine deficient (Zimmermann et al., 2008); iodized salt has since been supplemented into daily diets but still 30% of the world’s population suffered iodine deficiency in 2007 (de Benoist et al., 2008). Additional strategies for iodine supplementation have been considered (Smolen´ et al., 2014b). For example, increasing the iodine content of eatable plants was thought to be a safer and more efficient option than industrial iodized salt, as 75–85% of the iodine in human body comes from vegetable food under natural conditions (Weng et al., 2008). Increasing iodine in plants – iodine biofortification – is facilitated by using iodine-rich fertilisers.
⇑ Corresponding author. E-mail address: [email protected] (S. Pratt). http://dx.doi.org/10.1016/j.biortech.2015.06.063 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Using iodine rich fertilisers can lead to some fruit and vegetables having an iodine content of several mg kg1 (dry weight), e.g. carrot: 1 mg kg1, tomato: 6 mg kg1, soybean: 3.5 mg kg1 (Dai et al., 2004; Weng et al., 2014); leaf vegetables tend to have relatively higher iodine content, e.g. spinach: 65 mg kg1, celery: 50 mg kg1, Chinese cabbage: 80 mg kg1 (Nkemka and Murto, 2010; Singh and Ajay, 2011). Researchers have shown a linear relationship between iodine uptake rate and exogenous iodine (I, IO 3) level (0–10 mg L1) for cabbage and celery (Hong et al., 2009). Foliar fertilisation with KIO3 at 1 mg I L1 can result in lettuce leaves having an iodine content of 800 mg kg1 (Smolen´ et al., 2014a), which is comparable to some high iodine content seaweeds (Kupper et al., 2008). Algae have long been recognised as an important iodine accumulator in natural environments (Kupper et al., 2008). With the increasing interest in iodine biofortification, algae of high iodine content have been considered as a fertiliser. For example, ground dry kelp (0.2–0.3% iodine) was found to be an effective fertiliser
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for increasing iodine content in soil and improving iodine uptake by plants (Weng et al., 2008). The study showed kelp fertilisation of 40 mg kg1 (iodine to soil) could provide a residual iodine content of 25 mg kg1 in soil (free iodine) after 90 days plantation. Meanwhile, during the plantation, the iodine content of eatable plants was elevated to significant levels, e.g. cucumber: 6 mg kg1, aubergine: 10 mg kg1, radish: 8 mg kg1. Weng et al’s. (2008) study also found kelp as an iodine fertiliser can slow down iodine release in soil compared with potassium iodide (KI). Every year there are large amounts of waste algae generated from aquatic systems in many places around the world (Han et al., 2014). Algal fertiliser for iodine biofortification could represent a new opportunity for utilisation of waste algae with high iodine content. However, before applying to land, organic waste must be stabilised to avoid adverse environmental consequence (Polprasert, 1996). Stability refers to the rate or degree of organic matter decomposition, and can be expressed as a function of microbiological activity (Wu et al., 2000). It has been concluded that stabilisation increases the agricultural value of the material as a consequence of organic matter humification, reduces pathogens, and destroys some toxic compounds (Sanchez-Monedero et al., 2004). But there are currently no reports concerning iodine mobilisation during algae stabilisation. In this study, iodine-rich waste microalgae harvested from a groundwater holding pond was stabilised under both aerobic and anaerobic conditions. The organic carbon degradation and nutrient and iodine mobilisation were monitored. This is the first work considering iodine mobilisation during organic waste stabilisation. The outcomes are significant in the context of utilising the final products as fertiliser for iodine biofortification. 2. Methods In this work, ‘waste algae’ refers to algae harvested from a laboratory algal growth system (the laboratory growth system represents a groundwater holding pond, with iodine rich groundwater used as the growth medium). The waste algae was stabilised in (i) an aerobic environment, using activated sludge from a wastewater treatment plant as the inoculum for stabilisation, and (ii) an anaerobic environment, using digester sludge from a wastewater treatment plant as the inoculum for stabilisation. 2.1. Waste algae and inoculum for stabilisation Waste microalgae was collected from a lab scale production system that used iodine-rich groundwater (2.0 mg iodide L1) as the water source. The lab scale algal production system consisted of a 5 L large glass beaker illuminated with florescent light of 80 lmol m2 s1 at 25 °C using the groundwater as medium. After 4 months cultivation, the microalgae biofilm in the beaker was harvested and drained on a 500 lm pore size mesh plate. Then the mesh plate with algae was placed in a 4 °C fridge overnight for further drainage. To decompose the waste microalgae, wastewater treatment sludge was used as inoculum. For aerobic digestion, activated sludge was obtained from the aerobic zone at Luggage Point wastewater treatment plant (LPWWTP), Brisbane, Queensland, Australia. For anaerobic digestion, digester sludge was obtained from the same treatment plant. 2.2. Waste algae stabilisation Carbon emission was used as the measure of stabilisation. Stability refers to the rate or degree of organic matter decomposition, and can be expressed as a function of microbiological activity
(Wu et al., 2000), which in this case was determined by CO2 and CH4 production. The experimental set-ups for waste algae stabilisation are shown in Fig. 1. For aerobic digestion, 30 g wet algae, 15 g inoculum and 115 g distilled water were mixed in a 240 ml glass reactor. A magnetic stirrer was used for mixing. As shown in Fig. 1, input air flowed through two 240 ml glass bottles, the first containing saturated NaOH solution (200 ml), the second distilled water (200 ml), to ensure a CO2 free and water saturated air feed. After each reactor, there was another 240 ml glass bottle containing 100 ml NaOH solution (2 mol L1) for trapping the CO2 from exhaust gas. The four bottles were connected by tygon tubing in series for one way ventilation by compressed air. The aerobic stabilisation trial was repeated 4 times. The negative control, which was carried out in duplicate, contained additional water in place of the algae. Detailed set-up conditions are shown in Table 1. For anaerobic digestion, 30 g wet algae, 70 g inoculum and 60 g distilled water were mixed in 240 ml glass bottles, which were then sealed with butyl rubber stoppers. Before being sealed, the headspace was flushed with nitrogen gas. The anaerobic stabilisation trial was also repeated 4 times. The group of negative controls (triplicate) contained additional water in place of the algae. An active control experiment (duplicate) was also performed to verify the activity of the inoculum, by using 1.1 g cellulose as the substrate instead of algae.
2.3. Sampling and sample analysis The characteristics of the materials before and after stabilisation were analysed. During the stabilisation, a 1.5 ml liquid sample was taken from each reactor every one or two weeks. Total I, N, P and pH were measured in the liquid samples. To monitor waste microalgae biomass degradation during aerobic stabilisation, the NaOH solutions in the exhaust line were analysed for CO2 each time liquid was sampled from the reactors. After each sample, the exhaust gas traps were emptied and filled with fresh NaOH solution. For anaerobic stabilisation, a 6 ml gas sample from the headspace of each reactor was taken and measured while sampling the liquid. Carbon and nitrogen content of the algae was analysed by a FLASH 2000 CHNS/O Analyser. Compositions of gas samples were analysed by a Shimadzu GC-2014. For analysing CO2 produced by aerobic stabilisation, 1 ml NaOH solution from the exhaust gas traps was added to a sealed vacuum tube (12.5 ml capacity). Then each vacuum tube was filled with 4 ml of 4 M H2SO4 solution to ensure release of CO2 under acidic conditions. The CO2 content of the headspace of the vacuum tube was measured by GC. In the aquatic environment, the species of elemental iodine vary as a function of pH and redox conditions (Gottardi, 2001). Also, soluble organic iodine compounds may be present. So to quantify the total iodine mobilised into the liquid phase, tetramethylammonium hydroxide (TMAH) was used to pre-treat the samples to ensure all the iodine species were converted to inorganic iodide (Fecher et al., 1998). 0.1 ml of filtered liquid sample was mixed with 0.1 ml 25% TMAH solution. The mixture was then heated in a water bath at 90 °C for three hours. After cooling, the liquid samples were then diluted by distilled water to 1 ml and then analysed by a Dionex ICS-3000 Ion Chromatography system with an AS-18 column. For the detection of iodine content in algae, wet biofilm samples were rinsed with distilled water and dried at 110 °C overnight. 0.2 g dry algal biofilm solid was digested following a procedure reported by Ródenas de la Rocha et al. (2009). After digestion, the iodine content in the liquid was measured by the ion chromatography method. Nitrogen and phosphorus content were monitored by Flow Injection Analysis (FIA).
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Fig. 1. Experimental set-up for waste algae stabilisation.
Table 1 Set-up conditions of waste algae stabilisation. Process
Material
AG
NC
AC
Water (%)
VS (%)
Temp.(°C)
Air supply (ml min1)
Aerobic digestion
Algae Sludge Distilled water
30 g 15 g 115 g
0 15 g 145 g
0 0 0
91.9 98.9 100
7.8 0.9 0
25
35
Anaerobic digestion
Algae Sludge Distilled water Cellulose
30 g 70 g 60 g 0
0 70 g 90 g 0
0 70 g 90 g 1.1 g
91.8 97.8 100 0
7.8 2.1 0 100
37
0
AG: algae group. NC: negative control. AC: active control.
Table 2 Characteristics of initial materials used in the waste microalgae stabilisation experiments (±SD). Process
Group
Phase
Mass (g)
I content (ppm)
C (ppm)
N* (ppm)
P (ppm)
Total COD L + S (mg)
Aerobic
Algae
L S L S
0.02 ± 0.00 a: 350 ± 50 s: 14.3 0.00 ± 0.00 s: 14.3
0 a: 374000 s: 419000 0 s: 419000
6.89 ± 1.2 a: 65000 s: 84000 8.28 ± 1.49 s: 84000
9.09 ± 0.96 a: 6000 s: 12000 4.23 ± 1.24 s: 12000
2646 ± 243
NC
158.42 a: 2.44 ± 0.24 s: 0.17 ± 0.02 159.84 s: 0.17 ± 0.02
Algae
L S
NC
L S
157.12 a: 2.42 ± 0.24 s: 1.55 ± 0.07 158.53 s: 1.55 ± 0.07
0.43 ± 0.03 a: 350 ± 50 s: 51.7 0.30 ± 0.01 s: 51.7
0 a: 374000 s: 358000 0 s: 358000
304 ± 8.50 a: 65000 s: 57000 329 ± 7.10 s: 57000
29.6 ± 3.71 a: 6000 s: 20000 26.8 ± 0.75 s: 20000
Anaerobic
187.0 ± 0.94 4127 ± 275
1686 ± 33
NC: negative control; L: liquid phase; S: solid phase, all values in solid phase are on a dry weight basis. * Liquid phase nitrogen was the sum of nitrate-N and ammonium-N; a: values in algae; s: values in inoculum sludge.
2.4. Kinetic model Considering algae material consists of rapidly and slowly degradable components (Bai et al., 2014), a two-substrate model was applied in this study, with production of gas i (i = CH4, CO2) being described as follows:
Y i ðtÞ ¼ Y i;rapid ð1 ekrapid t Þ þ Y i;slow ð1 ekslow t Þ
ð1Þ
where Y i ðtÞ is the cumulative yield of a gas i at time t (ml g1 VSadded) (VSadded, volatile solid added to system); Y i;rapid is the maximum potential gas yield from the rapidly degradable substrate (ml g1 VSadded); krapid is hydrolysis rate constant for the degradation of rapidly degradable substrate (d1); Y i;slow is the maximum potential gas yield from the slowly degradable substrate (ml g1 VSadded); kslow is hydrolysis rate constant for the degradation of slowly degradable substrate (d1); and t is the time (d). The parameters were obtained from data fitting using the SigmaPlot 12.0 software.
The carbon emission from the aerobic and anaerobic stabilisations was determined from the sum of carbon dioxide and methane production (Aerobic: CO2-C; Anaerobic: CH4-C + CO2-C). i.e.,
X
EðtÞ ¼ M C
Y i ðtÞ
ð2Þ
i¼CH4 ;CO2
where EðtÞ is the cumulative carbon emission at time t (mg g1 VSadded); MC is the molar volumetric concentration of carbon (12/24.5 mg ml1, 25 °C). The carbon emission rate was used to determine the stability of organic wastes during a stabilisation process (Cabañas-Vargas et al., 2005; Kalamdhad et al., 2008). In this study, a model calculated value was used to indicate the carbon emission rate:
Erate ðtÞ ¼ EðtÞ0 where Erate ðtÞ is the (mg g1 VSadded d1).
ð3Þ carbon
emission
rate
at
time
t
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2.5. Mass balance calculation In a closed system, the elemental mass balance is:
Si C is ðxÞ þ Li C il ðxÞ þ Gi C ig ðxÞ ¼ Sf C fs ðxÞ þ Lf C fl ðxÞ þ Gf C fg ðxÞ
ð4Þ
where Si ; Li ; andGi are the initial mass of solid (dry basis), liquid, and gas phases (kg); Sf ; Lf ; and Gf are the final mass of solid (dry basis), liquid, and gas phases (kg); C is ðxÞ; C il ðxÞ; and C ig ðxÞ are the initial concentrations of the element in the solid (dry basis), liquid, and gas phases (mg kg1); and C fs ðxÞ; C fl ðxÞ; and C fg ðxÞ are the final concentrations in the solid (dry basis), liquid, and gas phases (mg kg1); In this study, mass balances were carried out in two steps. Firstly, a phosphorus mass balance was carried out to determine the actual initial dry solid mass Si in each reactor. P content in algae C is ðPÞ (dry basis) was measured as a highly repeatable value with very small standard deviations. Mass balances were then carried out for elemental C, I and N. The following parameters were assumed to be zero: C il ðCÞ, C ig ðCÞ, C ig ðIÞ, and C ig ðNÞ (CO2 and N2 in air were not considered). 3. Results and discussion 3.1. Characteristics of waste algae The harvested microalgae biofilm was mostly green and banded. Some of the biofilm that was harvested from the shade side of the cultivation beaker was brown. Under the microscope, the microalgae biofilm is characterised as a mat of green filamentous organisms with isolated cells of different morphologies mixed within the mat. The characteristics of the microalgae are shown in Table 2. The iodine content of the material was 350 ± 50 mg kg1. This value is much lower than that of kelp (2000–3000 mg kg1) (Weng et al., 2008), but still comparable to the iodine content in many reported seaweed species (Romaris-Hortas et al., 2011). Such an iodine content will potentially satisfy an efficient I-fertilisation dose, which has been reported to be 40 mg I per kg soil (Weng et al., 2008). Still, it is acknowledged that it would be important to study the in-field mineralisation of iodine in stabilised algae to confirm that the iodine would ultimately be available for plants. A feature of waste algae is that it is rich in nitrogen (Han et al., 2014). The nitrogen content (6.5%) of the waste microalgae in this study was higher than that of many organic wastes (Komilis et al., 2012). The content of phosphorous (0.6%) in waste microalgae was lower than the P content (1–2%) in some P-rich organic wastes and sludges (García-Romero et al., 2014; Rughoonundun et al., 2010; Wilkie and Mulbry, 2002), but higher than many reported waste algae materials (Bucholc et al., 2014; Ibrahim et al., 2014). These
Table 3 Kinetic parameters of biogas production using two-substrate model. Parameters
Fig. 2. Tracking carbon through the stabilisation processes. a: Carbon emission during aerobic and anaerobic stabilisation, (s) aerobic, (d) anaerobic; b: biogas production in anaerobic stabilisation, () CH4, (4) CO2; c: carbon mass balance, with liquid phase C determined as the difference between the initial C and the measured final solid and gas phase C, ( ) solid phase, (h) gas phase, (j) liquid phase (liquid phase carbon was obtained via calculation).
Y i;rapid (ml g1 VSadded) krapid (d1) Y i;slow (ml g1 VSadded) kslow (d1) Overall potential maximum biogas yield Y i;max (ml g1 VSadded) Overall potential maximum carbon emission Emax (mg g1 VSadded) Measured carbon emission after stabilisation (mg g1 VSadded) R2
Aerobic
Anaerobic
CO2
CH4
CO2
100.8 0.0183 111.4 0.0183 212.2
77.5 0.416 65.0 0.0318 142.5
37.1 0.0942 40.0 0.0102 77.1
103.9
69.8
37.8
87.6
69.6
31.2
0.983
0.997
0.999
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W. Han et al. / Bioresource Technology 193 (2015) 219–226 Table 4 Characteristics of final stabilisation products. Process
Group
Phase
Aerobic
Algae
L S L S
NC Anaerobic
Algae NC
L S L S
Mass (g) 158.6 1.20 ± 0.04* 159.9 0.05 ± 0.02 157.41 2.27 ± 0.12 158.70 1.03 ± 0.01
I content (ppm)
C (ppm)
N* (ppm)
1.37 ± 0.33 436.9 ± 92.4 0.01 ± 0.00 ND
ND 349800 ± 15700 ND 357700 ± 15600
273 ± 41.5 59300 ± 6100 70.3 ± 0.70 46000 ± 9900
45.9 ± 25.0 8900 ± 3200 20.3 ± 0.44 ND
1264 ± 126
3.05 ± 0.45 215.8 ± 17.2 0.45 ± 0.00 37.6 ± 1.1
ND 362600 ± 8600 ND 370800 ± 7800
992 ± 3.90 57100 ± 2000 596 ± 6.03 51400 ± 3900
90.2 ± 43.8 15300 ± 2100 84.6 ± 6.23 21100 ± 500
2102 ± 386
P (ppm)
Total COD L + S (mg)
83 ± 11
1340 ± 17
NC: negative control; ND: non detection. L: liquid phase; S: solid phase, all values in solid phase are on a dry weight basis. * Liquid phase nitrogen was the sum of nitrate-N and ammonium-N.
characteristics show the waste microalgae in this study to be potentially a useful fertiliser. 3.2. Stabilisation Algae was stabilised in both aerobic and anaerobic environments. Carbon emission, shown in Fig. 2a, was used as a measure of degradation. The kinetics differed but this was at least in part due to substantial differences in the substrate to inoculum ratios. It was assumed that stabilisation was ultimately first-order, which allowed for the determination of the extent of degradation and carbon emission rate. By the end of the trials (after 100 days), about 50% of the total CODadded of the algae was degraded (Tables 2 and 4). There was 21.9 ± 5.0% and 25.6 ± 1.9% (mean value ± SD) carbon emitted and the model calculated emission rates were 0.24 mg C g1 VSadded d1 (day100) and 0.10 mg C g1 VSadded d1 (day105) for the aerobic and anaerobic environments respectively, indicating that the material was ‘stable’ by the end of the trials (Cabañas-Vargas et al., 2005; Kalamdhad et al., 2008). Kinetic analysis was applied to help estimate the degradation potential of waste microalgae under the current stabilisation conditions. The calibrated parameters are shown in Table 3. The results show that the two-substrate model fits the measured values of carbon emission and biogas production (Fig. 2a and b). It seems there were no significant differences between rapidly and slowly degradable substrates in aerobic conditions, as the calibrated hydrolysis rate constants of both rapidly and slowly degradable substrates appeared to be the same value (0.0183 d1, Table 3). The rate constants (kCH4 ;rapid , kCH4 ;slow , kCO2 ;rapid ) for anaerobic stabilisation were higher than that for aerobic stabilisation (Table 3). Model simulation revealed Emax was 103.9 and 106.6 mg g1 VSadded in aerobic and anaerobic stabilisations respectively. The measured extent of carbon emission was 87.6 mg g1 VSadded (aerobic) after 100 days and 100.9 mg g1 VSadded (anaerobic) after 105 days. As such, most of the readily degradable substrates were degraded within 100 days. The methane production during anaerobic stabilisation was 140 ± 10 ml g1 VSadded or 130 ± 10 ml g1 CODadded (Fig. 2b). This yield was similar to some reported values for algae material that was stabilised anaerobically without pre-treatment (Allen et al., 2013; Nkemka and Murto, 2010). Carbon mass balance showed 172 ± 9.2 mg g1 VSadded (aerobic) and 182 ± 12 mg g1 VSadded (anaerobic) carbon remained in solid phase after degradation, which indicated 54 ± 7.3% (aerobic) and 52 ± 3.7% (anaerobic) of the total carbon was mobilised (Fig. 2c). 3.2.1. Mobilisation of iodine To examine the potential of using waste microalgae as an algae iodine fertiliser, it is important to understand the mobilisation of iodine during stabilisation. The degradation of the microalgae resulted in a total iodine level in the liquid of 0.56 ± 0.14
mg L1 g1 VSadded in the aerobic environment and 1.26 ± 0.19 mg L1 g1 VSadded in the anaerobic environment (Fig. 3a). The initial iodine content of the microalgae for both the aerobic and anaerobic stabilisations was similar (0.35 ± 0.05 mg g1 VSadded) (Fig. 3b). At the end of the stabilisations, 0.22 ± 0.05 mg g1 VSadded iodine remained in the solid phase in the aerobic environment while 0.19 ± 0.01 mg g1 VSadded iodine remained in the solid phase in the anaerobic environment (Fig. 3b). Iodine mobilisation in the aerobic environment (38 ± 5.0%) was less than in the anaerobic environment (50 ± 8.6%). Considering the degree of mobilised carbon was similar in both processes (around 50%, Fig. 2c), the results indicate iodine in waste microalgae is less readily mobilised during aerobic stabilisation. There was a strong linear correlation between the liquid phase iodine content and the emitted carbon in both environments (aerobic: R2 = 0.9901; anaerobic: R2 = 0.9914) (Fig. 3c). This indicated elemental iodine in waste microalgae was probably covalently bound to organic compounds rather than stored in the form of iodide. The latter had been reported in a few macroalgae species. From some macroalgae species, stored iodide can be released into the extracellular environment within a very short time (several hours) under specific environmental stimulations (normally oxidative stress) (Kupper et al., 2008; Nitschke et al., 2013). Organically bound iodine was reported in many living organisms, especially species in terrestrial water systems (Gribble, 2003; Li et al., 2013). Dehalogenation during the degradation of organic halocompounds can be an important process that causes release of halides (van Pée and Unversucht, 2003). A possible explanation for the lower level of iodine mobilisation in aerobic conditions is a difference in the mechanisms of dehalogenation under aerobic and anaerobic conditions. It has been shown that microbial catalysed dehalogenation under aerobic conditions is usually complicated and dependent on the level of NADH, while under anaerobic conditions an organic halocompound can serve as a terminal electron acceptor to form up a halo-respiration chain (van Pée and Unversucht, 2003). 3.2.2. Mobilisation of nutrients It is important to consider the mobilisation of nutrients if the stabilisation products are to be used as organic fertilisers. Liquid phase nitrogen was present as nitrate (NO 3 ) in aerobic conditions and as ammonium (NH+4) in anaerobic conditions (Fig. 4a and b). During stabilisation, decomposition of proteins results in release of ammonia. In the aerobic environment the mobilised ammonia is oxidised to NO 3 , which reduces pH (Hamoda and Ganczarczyk, 1980). The average pH in the aerobic environment was 6.8 at the beginning, with the pH eventually dropping to
