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Effects of sample size, dry ashing temperature and duration on determination of ash content in algae and other biomass
Algal Research 40 (2019) 101486
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Effects of sample size, dry ashing temperature and duration on determination of ash content in algae and other biomass
T
Keshun Liu Grain Chemistry and Utilization Laboratory, National Small Grains and Potato Germplasm Research Unit, United States Department of Agriculture, Agricultural Research Service (USDA-ARS), 1691 S. 2700 W., Aberdeen, ID 83210, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Dry ashing Ash content Biomass Algae Analytical method
Many reported methods on ash analysis by dry ashing vary greatly in ashing conditions, making results incomparable. The present study investigated effects of sample size (1, 4 g), ashing temperature (550, 600 °C) and duration (6, 16 h) on ash measurement of 13 algae and 4 non-algae (grains, soymeal, forage) samples under a factorial model. Results show that for most biomass both temperature and duration affected ash measurement significantly (p < .05). Ashing at 600 °C for 6 h or 550 °C overnight (about 16 h) gave values similar to ashing at 600 °C overnight, but ashing 6 h at 550 °C produced higher ash content than other combinations. Furthermore, for algae having higher ash content, sample size was also a determining factor when ashing at lower temperature (550 °C) and/or shorter duration (6 h), but the effect could be alleviated by ashing at 600 °C overnight. For comparable results, a standardized method of ashing 1 to 4 g samples at 600 °C overnight is proposed for all types of biomass.
1. Introduction Ash content or total ash represents the amount of total minerals present in a biomass. It is an important quality parameter. Many laboratories routinely conduct ash measurement as a part of biomass analysis for nutritional or compositional evaluation. In recent years, algae have gained attention due to their high production efficiency and versatile uses as animal feed, fertilizer, human food, and alternative feedstock for biofuel production [1–3]. Yet, algae are well known for their high levels and high variation in ash content [4–8]. Since high ash content in most algae diminishes their inclusion levels for food and feed [9] and poses major operational problems in biomass combustion systems for energy conversion [10,11], research has been conducted to remove ash from algae [8,11]. In eliminating variability in ash content, as with herbaceous biomass [12], algae are oftentimes assessed for ashfree dry weight. Therefore, an accurate and reliable measurement of ash content is critical in documenting quality of a biomass, whether it is a food, feed, industrial material, or renewable fuel feedstock. The ash content in algae and other biomass is commonly measured gravimetrically by burning samples in a muffle furnace at a high temperature for a specified duration. The process is known as dry oxidation or dry ashing. However, there has been large variation in ashing temperature and duration, as well as sample size (load), among reports for determining ash content in various biological materials. For non-algae biomass (such as food, feed and others), most researchers follow
standard methods, but the methods themselves vary with individual products. For example, for measuring ash content in cereals, pulses and by-products, AOAC International has an official method 923.03 [13], which is the same as ISO 2171 [14]. This method calls for igniting 3–5 g sample at 550 °C for 12–18 h. For animal feedstuffs, AOAC method 942.05 is usually used, which features heating a 2 g sample at 600 °C for 2 h [13]. For hard and soft woods, herbaceous materials, agricultural residues, wastepaper, and solid fractions upon processing, ASTM Standard Method E1755-01 [15] specifies burning 0.5 to 1.0 g samples at 575 ± 25 °C for a minimum of 3 h or until all the carbon is eliminated. Sluiter et al. [16] later modified the ASTM method by heating 0.5–2.0 g samples at 575 ± 25 °C for 24 ± 6 h. To determine ash content in algae, since there is no standard method available, dry ashing conditions vary considerably more among reports, with temperatures ranging 450–815 °C, durations 1–70 h, and sample size from 80 mg to several g, with most reports making no mention of sample size [4–8,11,17–22]. Because of the large variation in these factors, results among reports are difficult to compare. For ash analysis, it appears that most researchers are concerned only with ashing temperature and neglect the effect of ashing duration and sample size. Yet, it is hypothesized that although ashing temperature is an important factor, it is the combination of sample size, ashing temperature and duration that leads to incomplete or complete combustion. It is further hypothesized that low ashing temperature, short duration and large sample size can each lead to incomplete combustion, which in turn results in a higher ash
E-mail address: [email protected]. https://doi.org/10.1016/j.algal.2019.101486 Received 16 November 2018; Received in revised form 27 March 2019; Accepted 29 March 2019 Available online 17 April 2019 2211-9264/ Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2. Materials and methods
non-algae samples, an experiment with various combinations of ashing conditions (temperature and duration) and sample size was conducted in duplicate under a factorial design. Each factor had two levels: ashing temperature, 550 °C and 600 °C; ashing duration, 6 and 16 h (overnight); and sample load, 1 and 4 g, totaling eight combinations for temperature, duration, and sample size. The procedure for ash measurement followed the above proposed method, except for steps 3, 5 and 7, where changes were made to reflect the two levels for each factor.
2.1. Materials
2.5. Data analysis and statistical treatments
Thirteen algae samples were collected, in duplicate, from different sources and producers across the United States. They were named according to the order received. The names of producers/providers were omitted to avoid any possible negative impact on their products based on the results of this study. All samples were already dried by the providers prior to shipping, except for Algae 11, which was collected from a nearby waste water treatment facility. Upon collection, this sample was centrifuged at 3000 ×g for 10 min to remove extra water and dried in a forced air oven at 60 °C overnight. Non-algae materials included oat grain (cultivar Ajay), barley grain (cultivar Clearwater), defatted soy meal, and oat forage (cultivar CDC Dancer), representing grains (food), oilseed co-product (animal feed), and forage (herbaceous biomass). Barley grain, oat grain and forage were grown at the University of Idaho Experimental Station fields in Aberdeen, Idaho. Forage was harvested at a pre-flowering stage and dried in the forced air oven at 60 °C overnight. Defatted soy meal was obtained from the Archer Daniels Midland Co. (DeKalb, IL).
Replicate data on ash content were treated with JMP software, version 12 (JMP, a Business Unit of SAS Institute Inc., Cary, NC, USA). Samples were divided into algae and non-algae groups. Within each biomass group, analysis of variance (ANOVA) was conducted to determine the effects of sample type, sample size, ashing temperature, duration and their combinations on ash content. Tukey's honest significance test was then used for pair-wise comparisons of means, with a significance level of p < .05.
content than the true value. To test the hypotheses and provide valuable information to researchers dealing with ash measurement, the present study was conducted to investigate the effects of dry ashing temperature and duration, as well as sample size, on the ash measurement of algae and non-algae biomass. A part of the effort was to achieve the goal of providing a standardized method for measuring ash content in all biological materials.
3. Results In the present study, the effects of ashing temperature, duration, and sample size on ash measurement were investigated with 13 algae and 4 non-algae samples, under a factorial design. ANOVA results show that, for algae samples, all the three factors had significant effects (p < .05) on ash measurement (Table 1). Almost all the combinations also had significant interactive effects. Yet, for non-algae samples, only ashing temperature and ashing duration had significant effects on ash measurement. Sample size showed no significant effect. Most combinations of the three factors also had no interactive effects. As expected, for both groups, sample type also had a significant effect, indicating variation in ash content among samples. Careful examination of the actual ash content obtained by the 8 treatment combinations of the three factors (Table 2) indicates specific differences between algae and non-algae groups and among samples, with respect to the effects of ashing temperature, duration and sample size. For non-algae samples, when temperature was held at 600 °C, 6 h heating gave ash content (mean of 4 samples) no significantly different from that of 16 h. Yet, when ashing temperature was maintained at 550 °C, a 16 h duration was necessary to reach ash contents that were the same as those obtained at 600 °C. Ashing at 550 °C for 6 h gave mean values significantly higher than other treatment combinations. Sample size had no effect on ash content for each combination of ashing temperature and duration. When means were calculated under each of the two temperatures for individual non-algae samples (Table 2, column means), temperature had no effect for samples with lower ash content, but for samples with higher ash content (> 6%), 550 °C gave mean values significantly higher than 600 °C. Among the 13 algae samples, there was great variation in the ash content. The average values of four combinations of sample size and ashing duration at 600 °C for each sample (the far-right column, Table 2) ranged from 1.83 to 37.97%, dry matter basis. More importantly, based on the means of the 13 samples, 600 °C overnight ashing was required to achieve a constant ash content. With smaller sample size (1 g), ashing at 550 °C overnight or at 600 °C for 6 h produced ash values most close to the values obtained by ashing at 600 °C overnight. Ashing at 550 °C for 6 h with 1 or 4 g of sample and ashing at 550 °C for 16 h with 4 g sample size produced ash content significantly higher than other combinations. Similar to the observation with nonalgae samples, when means were calculated under each of the two temperatures for individual algae samples (Table 2, column means), for samples with lower ash content, ashing at 550 °C showed no significant difference from ashing at 600 °C, but for samples with higher ash content, ash values obtained at 550 °C were significantly higher than those
2.2. Proposed standard method for determining ash content The procedure for measuring ash content in all 17 samples (dried biomass) consisted of the following steps: 1) pre-conditioning porcelain crucibles in a muffle furnace under a vent hood at an ashing temperature of 600 °C for at least 30 min, to remove any combustible contaminants; 2) removing crucibles from the furnace and cooling to room temperature in a desiccator (about 1 h); 3) weighing each crucible to the nearest 0.1 mg, using gloves, tweezers, or tongs to prevent adding weight from hand moisture and contaminants; 4) weighing 1–4 g of each powdery sample (enough to produce 20 mg or more of ash) into the tared crucibles (as an exception, if a biomass had limited availability, its sample size could be < 1 g); 5) placing the crucibles with samples in the muffle furnace that had been set and pre-heated to a temperature of 600 °C under a vent hood; 6) pre-igniting samples by spontaneous auto combustion or with the aid of a lighter or match, while leaving the door slightly open to let smoke escape, and closing the door when no more smoke or flame appeared; 7) ashing the samples in the furnace overnight (about 16 h); 8) removing the crucibles with ashed samples from the muffle furnace for cooling to room temperature in a desiccator; 8) weighing each crucible with ash; and 9) calculating the total ash content as % sample mass (as is basis). 2.3. Measurement of moisture content For converting ash content to a dry matter basis, the moisture content of each sample was measured by drying the weighed samples (about 2 g each) in an oven at 105 °C ± 1 °C for 3 h, according to the AOAC Method 925.09 [13]. Note that sample moisture content could also be measured with the same muffle furnace prior to ash analysis, as a part of the overall procedure [15,20] instead of being carried out separately as shown in the present study. 2.4. The experiment for method development For method development in this study, using the 13 algae and 4 2
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Table 1 Effects of sample name, sample size, ashing temperature, ashing duration, and their interactions shown by analysis of variance. Source
For 13 algae samples Degree of freedom
Sample type Sample size Sample type ∗ Sample size Temperature Sample type ∗ Temperature Sample size ∗ Temperature Sample type ∗ Sample size ∗ Temperature Duration Sample type ∗ Duration Sample size ∗ Duration Sample type ∗ Sample size ∗ Duration Temperature ∗ Duration Sample type ∗ Temperature ∗ Duration Sample size ∗ Temperature ∗ Duration Sample type ∗ Sample size ∗ Temperature ∗ Duration
For four non-algae samples
Sum of square
12 1 12 1 12 1 12 1 12 1 12 1 12 1 12
34,170.239 13.756 65.605 42.021 81.932 2.823 52.421 19.846 43.627 1.242 2.584 0.003 2.379 0.018 2.760
F Ratio
Prob > F
Degree of freedom
28,940.050 139.802 55.564 427.070 69.391 28.686 44.397 201.704 36.950 12.618 2.188 0.029 2.015 0.178 2.337
< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0006 0.0173 0.8652 0.0299 0.6738 0.0108
3 1 3 1 3 1 3 1 3 1 3 1 3 1 3
Sum of square 920.005 0.002 0.166 0.268 0.085 0.011 0.025 0.135 0.077 0.029 0.028 0.008 0.046 0.007 0.022
F Ratio
Prob > F
49,017.930 0.289 8.865 42.806 4.535 1.762 1.335 21.587 4.092 4.619 1.491 1.295 2.462 1.088 1.179
< 0.0001 0.5948 0.0002 < 0.0001 0.0093 0.1937 0.2802 < 0.0001 0.0144 0.0393 0.2356 0.2636 0.0804 0.3048 0.3331
During the ashing process, water and volatiles are vaporized, organic substances are burned in the presence of oxygen in air, and minerals are converted to oxides, sulfates, phosphates, chlorides, and silicates. The mass of these compounds is weighed and % ash in a given sample is then calculated. Dry ashing is a safe and convenient method. It requires no added reagents, no blank for subtraction, and little attention once ignition begins. Many samples can be analyzed simultaneously. The method is also repeatable, as evidenced by data in Table 2, which shows mean of duplicate measurements for each data point, with an average relative standard deviation of 2.5% for all tests. The observation that for some samples ashing at 550 °C for 6 h gave higher ash values than ashing at 550 °C overnight or ashing at 600 °C for 6 h (Table 2) implies incomplete combustion under the conditions of lower temperature and/or shorter duration. For most samples, ashing at 600 °C overnight was found to give similar values to ashing at 600 °C for 6 h or 550 °C overnight, indicating that 600 °C overnight ashing did not cause significant loss of some minerals due to volatilization. The above
at 600 °C. Furthermore, under a specific ashing condition (a combination of temperature and duration), the effect of sample size was shown only in a few algae samples with very high ash content (> 14%) (Algae 3, 6, 7, 8 and 11). One exception was with Algae 5, which had a medium level of ash content but exhibited the most significant effect of sample size when ashing was conducted at 550 °C for 6 or 16 h, or at 600 °C for 6 h. Fortunately, the effect of sample size on ash determination at these combinations of ashing temperature and duration could be effectively alleviated by ashing at 600 °C overnight (Table 2). 4. Discussion Ash refers to the inorganic residue remaining after ignition or complete oxidation of organic matter in a biological material. Dry ashing has been the primary method to measure ash content in biomass. It is based on the principle that minerals are not destroyed by heating, since they have a lower volatility compared to other components.
Table 2 Total ash content of 13 algae and 4 non-algae biological materials as affected by sample size, ashing temperature and duration.a Ashing temperature (°C) Ashing time (hr) Sample size (g) Algae samples Algae 1 Algae 2 Algae 3 Algae 4 Algae 5 Algae 6 Algae 7 Algae 8 Algae 9 Algae 10 Algae 11 Algae 12 Algae 13 Meansc Non-algae samples Oat grain Barley grain Soy meal (defatted) Oat forage Meansc a b c
550
550
550
Meansb
550
6 1
6 4
16 1
16 4
6.14 7.03 39.20 1.88 11.38 33.71 40.27 15.24 7.31 6.57 31.82 12.89 10.67 17.24bc
6.14 7.59 40.10 1.89 20.36 34.15 40.50 15.21 7.44 6.51 31.63 13.25 10.83 18.12a
6.10 7.01 39.15 1.87 8.65 33.62 38.03 14.98 7.26 6.45 31.51 12.72 10.60 16.76de
6.05 6.97 39.17 1.91 15.54 33.64 39.46 14.93 7.17 6.38 31.20 12.76 10.71 17.37b
2.87 2.01 7.15 11.47 5.87ab
2.70 1.95 7.21 11.74 5.90a
2.81 1.95 7.06 11.47 5.82cd
2.63 1.95 6.85 11.46 5.72cd
600
600
600
Meansb
600
6 1
6 4
16 1
16 4
6.11o 7.15m 39.40a 1.88p 13.98i 33.78d 39.56a 15.09h 7.29m 6.48no 31.54f 12.90j 10.70k 17.37
6.08 7.01 38.04 1.81 10.53 33.12 36.43 14.37 7.24 6.52 30.89 12.59 10.67 16.56e
6.10 6.99 39.45 1.88 11.63 33.48 38.60 15.03 7.16 6.53 31.25 12.49 10.62 17.01cd
5.98 7.04 36.96 1.76 7.87 33.03 36.20 14.17 7.09 6.39 29.93 12.49 10.52 16.11f
6.09 6.96 37.42 1.90 7.94 32.95 36.60 14.07 7.16 6.40 30.27 12.45 10.63 16.22f
6.06o 7.00mn 37.97b 1.83p 9.49l 33.14e 36.95c 14.41i 7.16m 6.46no 30.58g 12.50j 10.61k 16.42
2.75e 1.96f 7.07c 11.53a 5.83
2.66 1.92 6.95 11.34 5.72cd
2.68 1.95 6.82 11.57 5.75bcd
2.64 1.95 6.92 11.16 5.67d
2.62 1.96 6.80 11.28 5.66d
2.65e 1.94f 6.87d 11.34b 5.70
Ash content was expressed as % dry matter basis for means of duplicate measurements. Average relative standard deviation for all tests, 2.5%. Column means within each group (algae or non-algae) for both temperatures (550 and 600 °C) with different letters differed significantly at p < .05. Row means within each group (algae or non-algae) for both temperatures (550 and 600 °C) with different letters differed significantly at p < .05. 3
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for 18 h [5], 550 °C for 3 h [11], 550 °C for 5 h [21], 550 °C for 70 h [4], 600 °C for 5 h [22], 600 °C for 16 h [7], and 815 °C for 1 h [19]. Based on the finding of the present study, some of these conditions might be inadequate for complete combustion of certain samples. The present study demonstrated a good and reasonable prospect of achieving the goal: to provide a standardized method for measuring ash content in all biomass. As shown in Table 2, for most biomass (algae and non-algae), overnight heating at either 550 °C or 600 °C would be suitable. Yet, ashing at 550 °C overnight or at 600 °C for 6 h was found insufficient to remove all organic matter from algae samples having high ash content. This was particularly true when sample size was larger (such as 4 g). Fortunately, Table 2 also shows that ashing at 600 °C overnight not only eliminated the effect of sample size on ash measurement for algae samples with high ash content, but also gave ash content that did not differ significantly from those obtained by ashing at 550 °C overnight or at 600 °C for 6 h, for algae samples with low ash content and non-algae samples. Therefore, the ashing condition of 600 °C overnight is recommended for ash determination in all types of biological samples. With regard to the effect of the sample size on dry ashing, in general, using a sample size larger than 4 g can overload crucibles when samples are very light (such as dry forage powder), while using a sample size smaller than 1 g can enlarge analytical errors for samples having ash content as low as 2%. Since the present study shows that dry ashing 1 or 4 g samples at 600 °C overnight gave the same ash content even for algae with high ash content (Table 2), 1 to 4 g sample load is recommended. However, there is an exception: using sample sizes smaller than 1 g for dry ashing may become necessary when availability of a biomass is rather limited. One example would be for small-scale laboratory cultures, where biomass is typically collected or concentrated by filtration with ash-free glass fiber filters (4, 17, 18). For all above considerations, a standard method of ashing 1–4 g biomass samples at 600 °C for 16 h (overnight) was thus proposed and described in the Materials and methods section.
observations also indicate that longer duration at a lower ashing temperature had the same effect as shorter duration at a higher ashing temperature in bringing combustion to completion, supporting the proposed hypotheses in the present study. There was a differential response between algae and non-algae sample groups toward the three treatment factors and their combinations (Table 1). Specifically, for algae samples with higher ash contents, there were not only strong effects of ashing temperature, ashing duration, and their combinations, but also a significant effect of sample size (Table 2). Yet, for non-algae samples and algae samples with lower ash content, sample size had no effect. These observations can be explained by the difference in ash composition between algae and non-algae samples and among algae samples. In the author's lab, ash from algae and other materials was recently characterized [7]. In general, ash in biomass can be divided into wet acid digestible ash and wet acid indigestible ash (WAIA). Being likely siliceous in nature, WAIA is an important contributor of algae ash since its content in algae positively correlates with total ash content. Upon microscopic examination, it was found that WAIA from algae consists of three siliceous materials: nondiatom cellular structures, diatom cell walls, and sandy particles, while WAIA from grains and forage mostly consists of non-diatom cellular structures [7]. The previous study concluded that high ash content of algae results mostly from contamination of diatoms and/or sandy particles of geologic origin. Relating to the present study, algae samples with higher ash content could produce ash with higher amounts of siliceous materials. During dry ashing, the ash produced on the top of a sample crucible could be heavy, form a natural and temporary cover for the remaining sample, and thus prevent the later from complete combustion. The protective effect of ash with higher amounts of silica could become more pronounced when a larger sample size was used for dry ashing. Fortunately, it could easily be abolished by ashing at a higher temperature for longer duration (such as 600 °C overnight, Table 2). Ash content is typically measured as a part of proximate analysis for algae and other biological samples. Although almost all reports on ash measurement use a muffle furnace, there is great variation in temperature and duration used for dry ashing. Pnakovic [23] compared two ashing conditions, 550 °C × 6 h and 815 °C × 6 h, in measuring ash content in wood samples of three tree species (10 g each) and found that on average the 550 °C × 6 h method gave 32% higher values than the 815 °C × 6 h method. The author pointed out the need for having certain standards for determining the ash content in wood material. The cited study also implies that, for biological materials, ashing at 815 °C can be too high to avoid measurable loss of some elements due to volatilization and decomposition. Xiao et al. [24] compared 2 h dry ashing at 500, 600 and 815 °C for non-algae biomass (rice straw, pine sawdust, tree leaf) and concluded that 600 °C is the optimal temperature. However, they did not look at the effect of ashing duration. Although the present study showed that for non-algae samples ashing at 550 °C overnight or at 600 °C for 6 h gave similar ash content (Table 2), it is not clear if ashing at 600 °C for 2 h is enough. For non-algae biomass, Sluiter et al. [16] specified ashing 0.5–2.0 g at 575 ± 25 °C for 24 ± 6 h. For algae biomass, van Wychen and Laurens [20] specified the same ashing temperature and duration ranges as Sluiter et al. [16] but with a lower sample size (100 mg). The two methods were all developed at National Renewable Energy Laboratory, U.S. Dept. of Energy. Golden, CO, as Laboratory Analytical Procedures (LAP). Based on results of the present study, the ashing condition and duration specified in LAP for non-algae biomass [16] and algae biomass [20] are mostly acceptable for complete combustion of organic matter but without measurable loss of volatiles and decomposition of inorganic compounds. Yet, for algae with very high ash content, a possible combination of ashing condition by the lowest end of temperature and duration ranges in LAP [20] (i.e., 550 °C for 18 h) may present a slight concern for incomplete combustion. Still, many other researchers used different ashing conditions for their studies with algae. Examples include 450 °C for 3 h [18], 450 °C for 24 h [6], 500 °C for 4 h [17], 530 °C
5. Conclusions The present study is the first to document that the interaction of ashing temperature and duration, rather than the temperature alone, influences ash measurement. It is also the first to show that, for algae samples with high ash content, sample size can be another determining factor. Therefore, when using dry ashing for ash analysis, one needs to choose a proper combination of ashing temperature, ashing time and sample size. Ashing 1–4 g samples at 600 °C overnight is proposed as a standard method and, thus, recommended for measuring ash content in all biomass (algae and non-algae biomass). Acknowledgements The author expresses appreciation to Mike Woolman, biological science technician of U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), for his assistance in conducting the experiments, Rick Barrows, Ph.D., retired research fish physiologist of USDA-ARS, and other individuals for helping and providing algae samples. This work was solely supported by the United States Federal Government appropriated fund for the project “Integrating the Development of New Feed Ingredients and Functionality and Genetic Improvement to Enhance Sustainable Production of Rainbow Trout” (No. 2050-21310-005-00-D), U.S. Department of Agriculture, Agricultural Research Service, Washington DC, USA. Declaration of authors' contributions to the work The author made substantial contributions to the conception and design of the study, acquisition of data, interpretation of data, drafting and developing the manuscript for submission. Before its submission, 4
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the paper was reviewed internally by a project team member, Dr. Thomas Welker, and approved by Research Leader, Dr. John M. Bonman.
diets for broiler chickens, J. Agric. Food Chem. 61 (2013) 7341–7348. [10] M. Hupa, Ash-related issues in fluidized-bed combustion of biomasses: recent research highlights, Energy Fuel 26 (2012) 4–14. [11] A. Robin, M. Sack, A. Israel, W. Frey, G. Müller, A. Golber, Deashing macroalgae biomass by pulsed electric field treatment, Bioresour. Technol. 255 (2018) 131–139. [12] R.R. Bakker, H.W. Elbersen, Managing ash content and quality in herbaceous biomass: an analysis from plant to product, Proceedings of the 14th European Biomass Conference Biomass for Energy, Industry and Climate Protection Held in Paris, France, Oct. 17–21, 2005. [13] AOAC (Association of Official Analytical Chemists), Official Methods of Analysis, 15th edition, Arlington, Virginia, USA, 1990. [14] ISO (the International Organization for Standardization), ISO 2171, Cereals, Pulses and By-products — Determination of Ash Yield by Incineration, 4th edition, (2007) (Geneva, Switzerland). [15] ASTM (American Society for Testing and Materials, International) E1755-01 “Standard method for the determination of ash in biomass” In 2003 Annual Book of ASTM Standards, vol. 11.05, (Philadelphia, PA). [16] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, Determination of Ash in Biomass: Laboratory Analytical Procedure. NREL/TP-510-42622, National Renewable Energy Laboratory, Golden, CO, 2008http://www.nrel.gov/docs/gen/ fy08/42622.pdf. [17] N. Nagao, T. Toda, K. Takaha, K. Hamaska, High ash content in net-plankton samples from shallow coastal water: possible source of error in dry weight measurement of zooplankton biomass, J. Oceanogr. 57 (2001) 105–107. [18] S.F. Sing, A. Isdepsky, M.A. Borowitzka, D.M. Lewis, Pilot-scale continuous recycling of growth medium for the mass culture of a halotolerant Tetraselmis sp. in raceway ponds under increasing salinity: a novel protocol for commercial microalgal biomass production, Bioresour. Technol. 161 (2014) 47–54. [19] H. Watanabe, D. Li, Y. Nakagawa, K. Tomishige, K. Kaya, M.M. Watanabe, Characterization of oil-extracted residue biomass of Botryococcus braunii as a biofuel feedstock and its pyrolytic behavior, Appl. Energy 132 (2014) 475–484. [20] S. van Wychen, L.M.I. Laurens, Determination of total solids and ash in algal biomass, Laboratory Analytical Procedure, National Renewable Energy Laboratory (NREL), U.S. Dept. of Energy, Golden, CO, 2015, www.nrel.gov/publications , Accessed date: 22 September 2018. [21] L. Yao, J.A. Cerde, S.-L. Lee, T. Wang, K.A. Harrata, Microalgae lipid characterization, J. Agric. Food Chem. 63 (2015) 1773–1787. [22] C. Ververis, K. Georghiou, D. Danielidis, D.G. Hatzinikolaou, P. Santas, R. Santas, V. Corleti, Cellulose, hemicelluloses, lignin and ash content of some organic materials and their suitability for use as paper pulp supplements, Bioresour. Technol. 98 (2007) 296–301. [23] L.D.-L. Pnakovic, Quantification of the ash content from biofuel - wood according to ISO 1171 (2003) and EN 14775 (2010), Annals of Warsaw University of Life Sciences – SGGW Forestry and Wood Technology 86 (2014) 86–91. [24] R. Xiao, X. Chen, F. Wang, G. Yu, The physicochemical properties of different biomass ashes at different ashing temperature, Renew. Energy 36 (2011) 244–249.
Conflict of interest statement The author declares that there was no conflict of interest or any potential financial or other interests that could be perceived to influence the outcomes of this research. Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. Declaration of authors The author agrees to the authorship and submission of the manuscript to Algal Research for peer review. References [1] J.A. Garrido-Cardenasa, F. Manzano-Agugliarob, F.G. Acien-Fernandezb, E. MolinaGrimab, Microalgae research worldwide, Algal Res. 35 (2018) 50–60. [2] E. Stephens, R. de Nys, I.L. Ross, B. Hankamer, Algae fuels as an alternative to petroleum, J. Pet. Environ. Biotechnol. 4 (2013) 148–155. [3] A.P. Matos, The impact of microalgae in food science and technology, J. Am. Oil Chem. Soc. 94 (2017) 1333–1350. [4] J.N.C. Whyte, Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves, Aquaculture 60 (1987) 231–241. [5] R.S.H. Rodde, K.M. Varum, B.A. Larsen, S.M. Myklestad, Seasonal and geographical variation in the chemical composition of the red alga Palmaria palmate (L.) Kuntze, Bot. Mar. 47 (2004) 125–133. [6] G.S. Costard, R.R. Machado, E. Barbario, R.C. Martino, S.O. Lourenço, Chemical composition of five marine microalgae that occur on the Brazilian coast, Int. J. Fish. Aquac. 4 (9) (2012) 191–201. [7] K. Liu, Characterization of ash in algae and other materials by determination of wet acid indigestible ash and microscopic examination, Algal Res. 25 (2017) 307–321. [8] J.E. Aston, B.D. Wahlen, R.W. Davis, A.J. Siccardi, L.M. Wendt, Application of aqueous alkaline extraction to remove ash from algae harvested from an algal turf scrubber, Algal Res. 35 (2018) 370–377. [9] R.E. Austic, A. Mustafa, B. Jung, S. Gatrell, X.G. Lei, Potential and limitation of a new defatted diatom microalgal biomass in replacing soybean meal and corn in
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Effects of sample size, dry ashing temperature and duration on determination of ash content in algae and other biomass
T
Keshun Liu Grain Chemistry and Utilization Laboratory, National Small Grains and Potato Germplasm Research Unit, United States Department of Agriculture, Agricultural Research Service (USDA-ARS), 1691 S. 2700 W., Aberdeen, ID 83210, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Dry ashing Ash content Biomass Algae Analytical method
Many reported methods on ash analysis by dry ashing vary greatly in ashing conditions, making results incomparable. The present study investigated effects of sample size (1, 4 g), ashing temperature (550, 600 °C) and duration (6, 16 h) on ash measurement of 13 algae and 4 non-algae (grains, soymeal, forage) samples under a factorial model. Results show that for most biomass both temperature and duration affected ash measurement significantly (p < .05). Ashing at 600 °C for 6 h or 550 °C overnight (about 16 h) gave values similar to ashing at 600 °C overnight, but ashing 6 h at 550 °C produced higher ash content than other combinations. Furthermore, for algae having higher ash content, sample size was also a determining factor when ashing at lower temperature (550 °C) and/or shorter duration (6 h), but the effect could be alleviated by ashing at 600 °C overnight. For comparable results, a standardized method of ashing 1 to 4 g samples at 600 °C overnight is proposed for all types of biomass.
1. Introduction Ash content or total ash represents the amount of total minerals present in a biomass. It is an important quality parameter. Many laboratories routinely conduct ash measurement as a part of biomass analysis for nutritional or compositional evaluation. In recent years, algae have gained attention due to their high production efficiency and versatile uses as animal feed, fertilizer, human food, and alternative feedstock for biofuel production [1–3]. Yet, algae are well known for their high levels and high variation in ash content [4–8]. Since high ash content in most algae diminishes their inclusion levels for food and feed [9] and poses major operational problems in biomass combustion systems for energy conversion [10,11], research has been conducted to remove ash from algae [8,11]. In eliminating variability in ash content, as with herbaceous biomass [12], algae are oftentimes assessed for ashfree dry weight. Therefore, an accurate and reliable measurement of ash content is critical in documenting quality of a biomass, whether it is a food, feed, industrial material, or renewable fuel feedstock. The ash content in algae and other biomass is commonly measured gravimetrically by burning samples in a muffle furnace at a high temperature for a specified duration. The process is known as dry oxidation or dry ashing. However, there has been large variation in ashing temperature and duration, as well as sample size (load), among reports for determining ash content in various biological materials. For non-algae biomass (such as food, feed and others), most researchers follow
standard methods, but the methods themselves vary with individual products. For example, for measuring ash content in cereals, pulses and by-products, AOAC International has an official method 923.03 [13], which is the same as ISO 2171 [14]. This method calls for igniting 3–5 g sample at 550 °C for 12–18 h. For animal feedstuffs, AOAC method 942.05 is usually used, which features heating a 2 g sample at 600 °C for 2 h [13]. For hard and soft woods, herbaceous materials, agricultural residues, wastepaper, and solid fractions upon processing, ASTM Standard Method E1755-01 [15] specifies burning 0.5 to 1.0 g samples at 575 ± 25 °C for a minimum of 3 h or until all the carbon is eliminated. Sluiter et al. [16] later modified the ASTM method by heating 0.5–2.0 g samples at 575 ± 25 °C for 24 ± 6 h. To determine ash content in algae, since there is no standard method available, dry ashing conditions vary considerably more among reports, with temperatures ranging 450–815 °C, durations 1–70 h, and sample size from 80 mg to several g, with most reports making no mention of sample size [4–8,11,17–22]. Because of the large variation in these factors, results among reports are difficult to compare. For ash analysis, it appears that most researchers are concerned only with ashing temperature and neglect the effect of ashing duration and sample size. Yet, it is hypothesized that although ashing temperature is an important factor, it is the combination of sample size, ashing temperature and duration that leads to incomplete or complete combustion. It is further hypothesized that low ashing temperature, short duration and large sample size can each lead to incomplete combustion, which in turn results in a higher ash
E-mail address: [email protected]. https://doi.org/10.1016/j.algal.2019.101486 Received 16 November 2018; Received in revised form 27 March 2019; Accepted 29 March 2019 Available online 17 April 2019 2211-9264/ Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2. Materials and methods
non-algae samples, an experiment with various combinations of ashing conditions (temperature and duration) and sample size was conducted in duplicate under a factorial design. Each factor had two levels: ashing temperature, 550 °C and 600 °C; ashing duration, 6 and 16 h (overnight); and sample load, 1 and 4 g, totaling eight combinations for temperature, duration, and sample size. The procedure for ash measurement followed the above proposed method, except for steps 3, 5 and 7, where changes were made to reflect the two levels for each factor.
2.1. Materials
2.5. Data analysis and statistical treatments
Thirteen algae samples were collected, in duplicate, from different sources and producers across the United States. They were named according to the order received. The names of producers/providers were omitted to avoid any possible negative impact on their products based on the results of this study. All samples were already dried by the providers prior to shipping, except for Algae 11, which was collected from a nearby waste water treatment facility. Upon collection, this sample was centrifuged at 3000 ×g for 10 min to remove extra water and dried in a forced air oven at 60 °C overnight. Non-algae materials included oat grain (cultivar Ajay), barley grain (cultivar Clearwater), defatted soy meal, and oat forage (cultivar CDC Dancer), representing grains (food), oilseed co-product (animal feed), and forage (herbaceous biomass). Barley grain, oat grain and forage were grown at the University of Idaho Experimental Station fields in Aberdeen, Idaho. Forage was harvested at a pre-flowering stage and dried in the forced air oven at 60 °C overnight. Defatted soy meal was obtained from the Archer Daniels Midland Co. (DeKalb, IL).
Replicate data on ash content were treated with JMP software, version 12 (JMP, a Business Unit of SAS Institute Inc., Cary, NC, USA). Samples were divided into algae and non-algae groups. Within each biomass group, analysis of variance (ANOVA) was conducted to determine the effects of sample type, sample size, ashing temperature, duration and their combinations on ash content. Tukey's honest significance test was then used for pair-wise comparisons of means, with a significance level of p < .05.
content than the true value. To test the hypotheses and provide valuable information to researchers dealing with ash measurement, the present study was conducted to investigate the effects of dry ashing temperature and duration, as well as sample size, on the ash measurement of algae and non-algae biomass. A part of the effort was to achieve the goal of providing a standardized method for measuring ash content in all biological materials.
3. Results In the present study, the effects of ashing temperature, duration, and sample size on ash measurement were investigated with 13 algae and 4 non-algae samples, under a factorial design. ANOVA results show that, for algae samples, all the three factors had significant effects (p < .05) on ash measurement (Table 1). Almost all the combinations also had significant interactive effects. Yet, for non-algae samples, only ashing temperature and ashing duration had significant effects on ash measurement. Sample size showed no significant effect. Most combinations of the three factors also had no interactive effects. As expected, for both groups, sample type also had a significant effect, indicating variation in ash content among samples. Careful examination of the actual ash content obtained by the 8 treatment combinations of the three factors (Table 2) indicates specific differences between algae and non-algae groups and among samples, with respect to the effects of ashing temperature, duration and sample size. For non-algae samples, when temperature was held at 600 °C, 6 h heating gave ash content (mean of 4 samples) no significantly different from that of 16 h. Yet, when ashing temperature was maintained at 550 °C, a 16 h duration was necessary to reach ash contents that were the same as those obtained at 600 °C. Ashing at 550 °C for 6 h gave mean values significantly higher than other treatment combinations. Sample size had no effect on ash content for each combination of ashing temperature and duration. When means were calculated under each of the two temperatures for individual non-algae samples (Table 2, column means), temperature had no effect for samples with lower ash content, but for samples with higher ash content (> 6%), 550 °C gave mean values significantly higher than 600 °C. Among the 13 algae samples, there was great variation in the ash content. The average values of four combinations of sample size and ashing duration at 600 °C for each sample (the far-right column, Table 2) ranged from 1.83 to 37.97%, dry matter basis. More importantly, based on the means of the 13 samples, 600 °C overnight ashing was required to achieve a constant ash content. With smaller sample size (1 g), ashing at 550 °C overnight or at 600 °C for 6 h produced ash values most close to the values obtained by ashing at 600 °C overnight. Ashing at 550 °C for 6 h with 1 or 4 g of sample and ashing at 550 °C for 16 h with 4 g sample size produced ash content significantly higher than other combinations. Similar to the observation with nonalgae samples, when means were calculated under each of the two temperatures for individual algae samples (Table 2, column means), for samples with lower ash content, ashing at 550 °C showed no significant difference from ashing at 600 °C, but for samples with higher ash content, ash values obtained at 550 °C were significantly higher than those
2.2. Proposed standard method for determining ash content The procedure for measuring ash content in all 17 samples (dried biomass) consisted of the following steps: 1) pre-conditioning porcelain crucibles in a muffle furnace under a vent hood at an ashing temperature of 600 °C for at least 30 min, to remove any combustible contaminants; 2) removing crucibles from the furnace and cooling to room temperature in a desiccator (about 1 h); 3) weighing each crucible to the nearest 0.1 mg, using gloves, tweezers, or tongs to prevent adding weight from hand moisture and contaminants; 4) weighing 1–4 g of each powdery sample (enough to produce 20 mg or more of ash) into the tared crucibles (as an exception, if a biomass had limited availability, its sample size could be < 1 g); 5) placing the crucibles with samples in the muffle furnace that had been set and pre-heated to a temperature of 600 °C under a vent hood; 6) pre-igniting samples by spontaneous auto combustion or with the aid of a lighter or match, while leaving the door slightly open to let smoke escape, and closing the door when no more smoke or flame appeared; 7) ashing the samples in the furnace overnight (about 16 h); 8) removing the crucibles with ashed samples from the muffle furnace for cooling to room temperature in a desiccator; 8) weighing each crucible with ash; and 9) calculating the total ash content as % sample mass (as is basis). 2.3. Measurement of moisture content For converting ash content to a dry matter basis, the moisture content of each sample was measured by drying the weighed samples (about 2 g each) in an oven at 105 °C ± 1 °C for 3 h, according to the AOAC Method 925.09 [13]. Note that sample moisture content could also be measured with the same muffle furnace prior to ash analysis, as a part of the overall procedure [15,20] instead of being carried out separately as shown in the present study. 2.4. The experiment for method development For method development in this study, using the 13 algae and 4 2
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Table 1 Effects of sample name, sample size, ashing temperature, ashing duration, and their interactions shown by analysis of variance. Source
For 13 algae samples Degree of freedom
Sample type Sample size Sample type ∗ Sample size Temperature Sample type ∗ Temperature Sample size ∗ Temperature Sample type ∗ Sample size ∗ Temperature Duration Sample type ∗ Duration Sample size ∗ Duration Sample type ∗ Sample size ∗ Duration Temperature ∗ Duration Sample type ∗ Temperature ∗ Duration Sample size ∗ Temperature ∗ Duration Sample type ∗ Sample size ∗ Temperature ∗ Duration
For four non-algae samples
Sum of square
12 1 12 1 12 1 12 1 12 1 12 1 12 1 12
34,170.239 13.756 65.605 42.021 81.932 2.823 52.421 19.846 43.627 1.242 2.584 0.003 2.379 0.018 2.760
F Ratio
Prob > F
Degree of freedom
28,940.050 139.802 55.564 427.070 69.391 28.686 44.397 201.704 36.950 12.618 2.188 0.029 2.015 0.178 2.337
< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0006 0.0173 0.8652 0.0299 0.6738 0.0108
3 1 3 1 3 1 3 1 3 1 3 1 3 1 3
Sum of square 920.005 0.002 0.166 0.268 0.085 0.011 0.025 0.135 0.077 0.029 0.028 0.008 0.046 0.007 0.022
F Ratio
Prob > F
49,017.930 0.289 8.865 42.806 4.535 1.762 1.335 21.587 4.092 4.619 1.491 1.295 2.462 1.088 1.179
< 0.0001 0.5948 0.0002 < 0.0001 0.0093 0.1937 0.2802 < 0.0001 0.0144 0.0393 0.2356 0.2636 0.0804 0.3048 0.3331
During the ashing process, water and volatiles are vaporized, organic substances are burned in the presence of oxygen in air, and minerals are converted to oxides, sulfates, phosphates, chlorides, and silicates. The mass of these compounds is weighed and % ash in a given sample is then calculated. Dry ashing is a safe and convenient method. It requires no added reagents, no blank for subtraction, and little attention once ignition begins. Many samples can be analyzed simultaneously. The method is also repeatable, as evidenced by data in Table 2, which shows mean of duplicate measurements for each data point, with an average relative standard deviation of 2.5% for all tests. The observation that for some samples ashing at 550 °C for 6 h gave higher ash values than ashing at 550 °C overnight or ashing at 600 °C for 6 h (Table 2) implies incomplete combustion under the conditions of lower temperature and/or shorter duration. For most samples, ashing at 600 °C overnight was found to give similar values to ashing at 600 °C for 6 h or 550 °C overnight, indicating that 600 °C overnight ashing did not cause significant loss of some minerals due to volatilization. The above
at 600 °C. Furthermore, under a specific ashing condition (a combination of temperature and duration), the effect of sample size was shown only in a few algae samples with very high ash content (> 14%) (Algae 3, 6, 7, 8 and 11). One exception was with Algae 5, which had a medium level of ash content but exhibited the most significant effect of sample size when ashing was conducted at 550 °C for 6 or 16 h, or at 600 °C for 6 h. Fortunately, the effect of sample size on ash determination at these combinations of ashing temperature and duration could be effectively alleviated by ashing at 600 °C overnight (Table 2). 4. Discussion Ash refers to the inorganic residue remaining after ignition or complete oxidation of organic matter in a biological material. Dry ashing has been the primary method to measure ash content in biomass. It is based on the principle that minerals are not destroyed by heating, since they have a lower volatility compared to other components.
Table 2 Total ash content of 13 algae and 4 non-algae biological materials as affected by sample size, ashing temperature and duration.a Ashing temperature (°C) Ashing time (hr) Sample size (g) Algae samples Algae 1 Algae 2 Algae 3 Algae 4 Algae 5 Algae 6 Algae 7 Algae 8 Algae 9 Algae 10 Algae 11 Algae 12 Algae 13 Meansc Non-algae samples Oat grain Barley grain Soy meal (defatted) Oat forage Meansc a b c
550
550
550
Meansb
550
6 1
6 4
16 1
16 4
6.14 7.03 39.20 1.88 11.38 33.71 40.27 15.24 7.31 6.57 31.82 12.89 10.67 17.24bc
6.14 7.59 40.10 1.89 20.36 34.15 40.50 15.21 7.44 6.51 31.63 13.25 10.83 18.12a
6.10 7.01 39.15 1.87 8.65 33.62 38.03 14.98 7.26 6.45 31.51 12.72 10.60 16.76de
6.05 6.97 39.17 1.91 15.54 33.64 39.46 14.93 7.17 6.38 31.20 12.76 10.71 17.37b
2.87 2.01 7.15 11.47 5.87ab
2.70 1.95 7.21 11.74 5.90a
2.81 1.95 7.06 11.47 5.82cd
2.63 1.95 6.85 11.46 5.72cd
600
600
600
Meansb
600
6 1
6 4
16 1
16 4
6.11o 7.15m 39.40a 1.88p 13.98i 33.78d 39.56a 15.09h 7.29m 6.48no 31.54f 12.90j 10.70k 17.37
6.08 7.01 38.04 1.81 10.53 33.12 36.43 14.37 7.24 6.52 30.89 12.59 10.67 16.56e
6.10 6.99 39.45 1.88 11.63 33.48 38.60 15.03 7.16 6.53 31.25 12.49 10.62 17.01cd
5.98 7.04 36.96 1.76 7.87 33.03 36.20 14.17 7.09 6.39 29.93 12.49 10.52 16.11f
6.09 6.96 37.42 1.90 7.94 32.95 36.60 14.07 7.16 6.40 30.27 12.45 10.63 16.22f
6.06o 7.00mn 37.97b 1.83p 9.49l 33.14e 36.95c 14.41i 7.16m 6.46no 30.58g 12.50j 10.61k 16.42
2.75e 1.96f 7.07c 11.53a 5.83
2.66 1.92 6.95 11.34 5.72cd
2.68 1.95 6.82 11.57 5.75bcd
2.64 1.95 6.92 11.16 5.67d
2.62 1.96 6.80 11.28 5.66d
2.65e 1.94f 6.87d 11.34b 5.70
Ash content was expressed as % dry matter basis for means of duplicate measurements. Average relative standard deviation for all tests, 2.5%. Column means within each group (algae or non-algae) for both temperatures (550 and 600 °C) with different letters differed significantly at p < .05. Row means within each group (algae or non-algae) for both temperatures (550 and 600 °C) with different letters differed significantly at p < .05. 3
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for 18 h [5], 550 °C for 3 h [11], 550 °C for 5 h [21], 550 °C for 70 h [4], 600 °C for 5 h [22], 600 °C for 16 h [7], and 815 °C for 1 h [19]. Based on the finding of the present study, some of these conditions might be inadequate for complete combustion of certain samples. The present study demonstrated a good and reasonable prospect of achieving the goal: to provide a standardized method for measuring ash content in all biomass. As shown in Table 2, for most biomass (algae and non-algae), overnight heating at either 550 °C or 600 °C would be suitable. Yet, ashing at 550 °C overnight or at 600 °C for 6 h was found insufficient to remove all organic matter from algae samples having high ash content. This was particularly true when sample size was larger (such as 4 g). Fortunately, Table 2 also shows that ashing at 600 °C overnight not only eliminated the effect of sample size on ash measurement for algae samples with high ash content, but also gave ash content that did not differ significantly from those obtained by ashing at 550 °C overnight or at 600 °C for 6 h, for algae samples with low ash content and non-algae samples. Therefore, the ashing condition of 600 °C overnight is recommended for ash determination in all types of biological samples. With regard to the effect of the sample size on dry ashing, in general, using a sample size larger than 4 g can overload crucibles when samples are very light (such as dry forage powder), while using a sample size smaller than 1 g can enlarge analytical errors for samples having ash content as low as 2%. Since the present study shows that dry ashing 1 or 4 g samples at 600 °C overnight gave the same ash content even for algae with high ash content (Table 2), 1 to 4 g sample load is recommended. However, there is an exception: using sample sizes smaller than 1 g for dry ashing may become necessary when availability of a biomass is rather limited. One example would be for small-scale laboratory cultures, where biomass is typically collected or concentrated by filtration with ash-free glass fiber filters (4, 17, 18). For all above considerations, a standard method of ashing 1–4 g biomass samples at 600 °C for 16 h (overnight) was thus proposed and described in the Materials and methods section.
observations also indicate that longer duration at a lower ashing temperature had the same effect as shorter duration at a higher ashing temperature in bringing combustion to completion, supporting the proposed hypotheses in the present study. There was a differential response between algae and non-algae sample groups toward the three treatment factors and their combinations (Table 1). Specifically, for algae samples with higher ash contents, there were not only strong effects of ashing temperature, ashing duration, and their combinations, but also a significant effect of sample size (Table 2). Yet, for non-algae samples and algae samples with lower ash content, sample size had no effect. These observations can be explained by the difference in ash composition between algae and non-algae samples and among algae samples. In the author's lab, ash from algae and other materials was recently characterized [7]. In general, ash in biomass can be divided into wet acid digestible ash and wet acid indigestible ash (WAIA). Being likely siliceous in nature, WAIA is an important contributor of algae ash since its content in algae positively correlates with total ash content. Upon microscopic examination, it was found that WAIA from algae consists of three siliceous materials: nondiatom cellular structures, diatom cell walls, and sandy particles, while WAIA from grains and forage mostly consists of non-diatom cellular structures [7]. The previous study concluded that high ash content of algae results mostly from contamination of diatoms and/or sandy particles of geologic origin. Relating to the present study, algae samples with higher ash content could produce ash with higher amounts of siliceous materials. During dry ashing, the ash produced on the top of a sample crucible could be heavy, form a natural and temporary cover for the remaining sample, and thus prevent the later from complete combustion. The protective effect of ash with higher amounts of silica could become more pronounced when a larger sample size was used for dry ashing. Fortunately, it could easily be abolished by ashing at a higher temperature for longer duration (such as 600 °C overnight, Table 2). Ash content is typically measured as a part of proximate analysis for algae and other biological samples. Although almost all reports on ash measurement use a muffle furnace, there is great variation in temperature and duration used for dry ashing. Pnakovic [23] compared two ashing conditions, 550 °C × 6 h and 815 °C × 6 h, in measuring ash content in wood samples of three tree species (10 g each) and found that on average the 550 °C × 6 h method gave 32% higher values than the 815 °C × 6 h method. The author pointed out the need for having certain standards for determining the ash content in wood material. The cited study also implies that, for biological materials, ashing at 815 °C can be too high to avoid measurable loss of some elements due to volatilization and decomposition. Xiao et al. [24] compared 2 h dry ashing at 500, 600 and 815 °C for non-algae biomass (rice straw, pine sawdust, tree leaf) and concluded that 600 °C is the optimal temperature. However, they did not look at the effect of ashing duration. Although the present study showed that for non-algae samples ashing at 550 °C overnight or at 600 °C for 6 h gave similar ash content (Table 2), it is not clear if ashing at 600 °C for 2 h is enough. For non-algae biomass, Sluiter et al. [16] specified ashing 0.5–2.0 g at 575 ± 25 °C for 24 ± 6 h. For algae biomass, van Wychen and Laurens [20] specified the same ashing temperature and duration ranges as Sluiter et al. [16] but with a lower sample size (100 mg). The two methods were all developed at National Renewable Energy Laboratory, U.S. Dept. of Energy. Golden, CO, as Laboratory Analytical Procedures (LAP). Based on results of the present study, the ashing condition and duration specified in LAP for non-algae biomass [16] and algae biomass [20] are mostly acceptable for complete combustion of organic matter but without measurable loss of volatiles and decomposition of inorganic compounds. Yet, for algae with very high ash content, a possible combination of ashing condition by the lowest end of temperature and duration ranges in LAP [20] (i.e., 550 °C for 18 h) may present a slight concern for incomplete combustion. Still, many other researchers used different ashing conditions for their studies with algae. Examples include 450 °C for 3 h [18], 450 °C for 24 h [6], 500 °C for 4 h [17], 530 °C
5. Conclusions The present study is the first to document that the interaction of ashing temperature and duration, rather than the temperature alone, influences ash measurement. It is also the first to show that, for algae samples with high ash content, sample size can be another determining factor. Therefore, when using dry ashing for ash analysis, one needs to choose a proper combination of ashing temperature, ashing time and sample size. Ashing 1–4 g samples at 600 °C overnight is proposed as a standard method and, thus, recommended for measuring ash content in all biomass (algae and non-algae biomass). Acknowledgements The author expresses appreciation to Mike Woolman, biological science technician of U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), for his assistance in conducting the experiments, Rick Barrows, Ph.D., retired research fish physiologist of USDA-ARS, and other individuals for helping and providing algae samples. This work was solely supported by the United States Federal Government appropriated fund for the project “Integrating the Development of New Feed Ingredients and Functionality and Genetic Improvement to Enhance Sustainable Production of Rainbow Trout” (No. 2050-21310-005-00-D), U.S. Department of Agriculture, Agricultural Research Service, Washington DC, USA. Declaration of authors' contributions to the work The author made substantial contributions to the conception and design of the study, acquisition of data, interpretation of data, drafting and developing the manuscript for submission. Before its submission, 4
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the paper was reviewed internally by a project team member, Dr. Thomas Welker, and approved by Research Leader, Dr. John M. Bonman.
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