Microbotanical analysis: Starch residues Experimentation with starch extraction techniques

In document Agriculture in Tongan Prehistory: An Archaeobotanical Perspective (Page 144-154)

As emphasised within Chapter 3, a number of previous technical studies of starch residue extraction and processing from sediments have highlighted issues with current protocols, such as sampling strategies, contamination and the use of destructive chemicals (Barton et al. 1998; Coil 2003; Crowther 2009; Korstanje 2003; Parr 2002; Therin and Lentfer, in Torrence 2006b; Torrence 2006b; Torrence and Therin, in Torrence 2006b). New protocols are constantly developed to address the effects of different taphonomic, environmental and laboratory conditions. In the current study, several experiments were designed to solve issues that arose during the first round of laboratory processing, especially potential contamination from the chemicals and equipment used. This experimentation sought to deal with this potential contamination in the laboratory setting, as well as issues with the removal of micro-charcoal that can inhibit viewing of starch granules on glass slides and the recycling of heavy liquid. The results of these experiments enabled the development of a revised protocol that suited the nature of the archaeological deposits being sampled and the laboratory equipment available at the ANU.

Experiment One: Potential for starch contamination from Calgon, de-ionised water, filter mesh, LST and Glycerol

The risk of contamination during post-excavation processing has been outlined in previous research by Loy and Barton (in Torrence 2006b), Laurence (2013) and most recently Crowther (2014). Experiments were designed to investigate the potential for contamination from materials

127 used to process the archaeological sediments. To do this, each material was independently tested for the presence of starch, and any observed starch was recorded and counted.

Calgon

Firstly, powdered Calgon or sodium hexametaphosphate was tested by mixing the powder with de-ionised water to produce four 500ml samples of 5% Calgon. A magnetic mixer was added to each of the samples, and these were then placed on a hotplate set to 20˚C to allow the Calgon to dissolve into the de-ionised water. Once this had occurred, the samples were each filtered through a 10µm laboratory-grade-filtration mesh. The mesh was then cut into three parts, mounted onto slides, and covered with a cover-slip. Mountant was not added to the samples, as these were constructed only as temporary slide mounts. The slides were then viewed using light microscopy in both brightfield and polarised light.

The results of this experiment suggested relatively high numbers of starch contaminants from powdered Calgon. Sample 1 had a total of 35 grains, Sample 2 contained 12 grains, Sample 3 had 69 grains, and Sample 4 had 37 grains. The predominant starch types encountered were taxonomically identified as wheat (Triticum spp.), and maize (Zea mays). It is possible that contamination of the Calgon occurred in the laboratory storage for the Department of Archaeology and Natural History, as the bag had been left open for a time. It is also plausible that some contamination can occur even within laboratory-grade Calgon during manufacture, as many companies produce both Calgon and powdered maize (along with other dehydrated or milled plant products).

To resolve this issue and enable Calgon to be used as a deflocculant during processing of the archaeological samples, the 5% Calgon/de-ionised water mix was filtered through a 5µm laboratory filtration mesh to remove any potential starch contaminants. The filtered mix was then always covered unless being added to soil samples in starch extraction processes to break down clay particles.

De-ionised water

Four 500ml samples of de-ionised water were processed in a similar manner to the Calgon. The beakers were poured through a circle of 10µm filtration mesh and the filter paper was then cut into three parts and mounted on slides as temporary mounts. No starch was observed within these samples during light microscopy, and so it was concluded that de-ionised water could not be a source of starch contamination when used in extracting starch from archaeological sediments.

Filtration mesh

Four circles of 10µm mesh laboratory-grade filtration mesh were dampened with de-ionised water, after previous experimentation revealed that this was not a potential source of starch contamination. These circles were then each cut into three parts, placed onto slides, and then

128 covered with a coverslip. Light microscopy revealed that very little starch can be found on filtration paper, with only one to two grains observed on any of the samples (Sample 2), and therefore these are also not a significant source of contamination during processing of archaeological sediments.

Lithium hexametaphosphate (LST)

Two samples of unused and two samples of recycled LST heavy liquid at 2.0sg were also tested for starch contamination. These were filtered through individual rounds of 10µm filtration mesh, and cut into strips before being placed on slides as temporary mounts. These slides were then observed using light microscopy and any observed starch morphotypes were recorded. No starch was observed within any of these samples, indicating that these are not sources of starch contamination during processing.

Glycerol

Glycerol was the mountant chosen to create permanently mounted slides for both the comparative collection and the starch extracted from archaeological sediments. It has a high refractive index and ensures starch is preserved and protected from enzymatic attack, while also allowing starch granules to be rolled. To test whether the Glycerol used in the Palynological Laboratory in the Department of Archaeology and Natural History could be a potential source of starch contamination, four slides were produced with 100µl of Glycerol on each. These slides were covered with a cover-slip, and then observed using light microscopy. Three wheat starch granules were found in total on these four slides, and so this material is a potential source of starch contamination, but the contamination is very small.

Conclusions

These experiments assessed the amount and types of starch within a number of standard or regularly employed chemicals and materials used to extract starch residues. Of the five materials tested, only three contained any visible starch. Glycerol and filtration paper had very small amounts of starch, with less than three granules observed in any one sample. Based on these results it was decided that the use of glycerol and filtration paper within the current protocol was acceptable and required no further processing. In contrast, the test involving unfiltered Calgon highlighted that this material could be a significant source of contamination from modern wheat and maize starch. Up to 69 starch grains were observed in each of the four samples. These findings indicate that a major revision of the deflocculation process was required to enable archaeological sediments to be processed without the risk of contamination. In light of this, it was decided that the pre-mixed Calgon needed to be filtered through a 5µm filtration mesh prior to use. All other materials were acceptable for use within the revised laboratory protocol. Experiment Two: Removal of charcoal from samples

The soil samples collected from Talasiu, Leka and Heketa were taken from shell middens of various densities, and it was noted that there were high concentrations of microcharcoal present

129 in the soil matrix. Microcharcoal can make light microscopy difficult by obscuring the visibility of any potential starch grains, and so needs to be either removed or dispersed in starch extraction. Several experiments were carried out to establish whether particular methods were effective in this, and also to gauge the effect these methods had on starch residues. These replicated and built on experiments carried out by Crowther (2009:62-82).

Lithium hexametaphosphate (LST)

One method tested for the removal of charcoal was to use Heavy Liquid to remove light fractions of material from soil samples. Wood charcoal has a specific gravity (sg) or ratio of density of 0.4sg, which enables it to float on water during archaeobotanical flotation techniques. However, the microcharcoal observed in this experiment has instead settled in water during Step Two of the methodology outlined here. It was hypothesised that this microcharcoal may be able to be removed using a heavy liquid with a specific gravity heavier than water (1.0sg), but lighter than the specific gravity of starch (1.7sg).

One archaeological sediment sample (Talasiu Spit 18) was selected to be sub-sampled and processed to the point where Heavy Liquid in the form of lithium heteropolytungstate (LST) was added using the methodology outlined below. Thirty millilitres of LST at 2.0sg was added to the sample, and centrifuged at 1500rpm for 30mins. This was decanted through a 5µm filtration mesh, enabling the LST to be separated from the extracted material and thus easily recycled. The mesh was then placed in the top of a Falcon tube and de-ionised water was used to wash the material caught on the mesh into the tube. All material lighter than 2.0sg and larger than 5µm was therefore retained. Another LST solution at 1.2sg was then added, and the process repeated so that a fraction that was lighter than 1.2sg was separated. The heavy fraction was also retained as this should contain any starch residues.

The results of this experiment indicate that microcharcoal cannot be separated using LST set at 1.2sg. Most of the charcoal examined tended to become waterlogged when exposed to liquid over a sustained period of time, and became too dense to float. The differential specific gravities of microcharcoals are probably a result of differing wood densities before charring, particle size, and density as a result of water absorption. These changes probably occur either in the soil or during Step One and Step Two.

Hydrogen peroxide (H2O2)

The second method tested in an attempt to remove charcoal from the archaeological sediments was the addition of hydrogen peroxide or H2O2. Talasiu Spit 18 was again selected to be sub-

sampled for this experiment. The sample was processed until the point where it was reduced to allow slide preparation. The sample was placed into a thin glass centrifuge tube under the fume hood, and 10% hydrogen peroxide was added to the sample one drop at a time to gauge any chemical reaction. There was no immediate reaction, nor to a higher percentage dilution of 30%

130 added in the same manner. There was only a very small reduction in the amount of charcoal observed within the sample.

The final test in this experiment involved the application of 30% hydrogen peroxide and then the sample was placed in a beaker of water on a hotplate to stimulate a stronger chemical reaction. The water was warmed to 30˚C, and the sample was observed for 1.5 hours within which time there was still no reaction. A slide was prepared after the final test in this experiment to observe any effects of these chemicals on starch morphology. It was clear that most starch had either gelatinised or dissolved. Very low numbers of starch in native condition were observed in this test sample.

Simple dispersal

The final method tested in this experiment was not to remove the charcoal, but to allow simple dispersal within the sample. Rather than preparing only one or two slides from the reduced samples after processing, three to four slides were constructed. This allowed any micro-charcoal present in the samples to be dispersed over a larger number of slides, reducing the possibility of these charcoal particles obscuring any starch granules present in the samples.

Conclusions

The use of chemical agents to remove microcharcoal from archaeological sediments was not successful for a variety of reasons. Heavy Liquid separation using LST at 1.2sg was not able to float off micro-charcoal, due to the varying densities of wood charcoal. Some microcharcoal was removed during this process, but high concentrations remained in the heavy fraction that also contained the extracted starch grains. Similarly, 10% and 30% hydrogen peroxide proved to be ineffective in dissolving charcoal, even when heat was added in an attempt to produce a stronger reaction. In addition, such concentrations of hydrogen peroxide affected starch morphology and preservation, rendering the use of these chemicals unacceptable.

Experiment Three: Removal of supernatant and recycling of heavy liquid

A simple experiment was carried out during the first phase of laboratory processing of the archaeological soil samples to allow the separation of the supernatant after heavy liquid in the form of lithium heteropolytungstate (LST) was added. Current methodologies (Horrocks 2004; Therin and Lentfer, in Torrence 2006b) either pipette or decant the supernatant into a separate tube, leaving the heavy residue behind in the original centrifuge tube to either be kept for a second phase of heavy liquid separation or to be discarded. Experimentation was conducted to ease recycling of heavy liquid for use within other samples and reduce inter-sample contamination.

During this experiment six samples from one spit (level) at Talasiu (TO-Mu-2) were processed up to the stage prior to heavy liquid separation. Exactly 30ml of LST at 2.0sg was added to all four of these samples within centrifuge tubes which were then placed into the

131 centrifuge and spun at 1500rpm for 15mins. At this point two samples were chosen to attempt pipetting off the supernatant, two samples were selected for decanting, and two samples were chosen to test a new method which involved decanting the supernatant through a 5µm mesh (Janelle Stevenson, pers.com). The reason for attempting this new method was to reduce the amount of non-starch material within the sample after heavy liquid processing and to ease the process of recycling the heavy liquid through separating the starch from the LST immediately. Each set of samples were then processed accordingly. Individual techniques for separating the supernatant were assessed for ease of use in terms of the time it took to carry out the separation, the efficiency of the separation in terms of the amount of heavy residue within the supernatant, and the subsequent steps needed to remove and recycle the heavy liquid.

Pipette Technique

Pipetting the supernatant off the top of the heavy residue which had sunk to the bottom of the centrifuge tube proved to be a very efficient technique for separating the light and heavy fractions. Very little of the heavy residue was accidentally added to the light fraction. However, the method was also very time-consuming as only small amounts of the supernatant could be removed at a time, taking around 5 minutes. The heavy liquid then needed to be removed from the light fraction through various steps involving the dilution of the sample to wash the heavy liquid and retain starch residues. The heavy liquid then needed to be passed through a glass mesh to remove any contaminants. To increase the specific gravity of the heavy liquid, it was put inside a glass beaker on a magnetic stirrer at low heat and monitored until enough water had evaporated to return the LST to 2.0sg. This was again a time-consuming process, taking around eight hours to enable the heavy liquid to be reused.

Decanting Technique

Decanting the supernatant into a new centrifuge tube took less time to separate the heavy and light fractions—approximately 1 minute— but was less effective. There was some mixing of the two fractions as the last of the supernatant was carefully poured into the second tube. This meant that the final slides had more organic material than those made from the pipette samples. The same process was then used to recycle the heavy liquid through dilution, sieving and evaporating water to return the LST to 2.0sg.

Sieving Technique

Pouring the supernatant through the 5µm mesh was a time-consuming process as small amounts of organic residue from the top of the heavy fraction was inter-mixed with the light fraction, as seen within the decanting technique, and combined with charcoal to clog the mesh. A pump had to be used to slowly allow the supernatant to pass through the mesh, taking around 10 minutes. The light fraction remained on the 5µm mesh, and was then put into the top of a centrifuge tube and washed off into the tube using de-ionised water. Only one wash was required to remove any residual LST from the sample before the sample could be reduced for slide construction.

132 Despite being more time-consuming in terms of separating the supernatant from the heavy fraction, the technique was very effective at separating the heavy liquid from the light fraction quickly without any dilution, enabling the LST to be used immediately after only one further step of sieving through a glass filter paper.

Conclusions

Both pipetting and decanting the supernatant off from above the heavy fraction were more time- effective methods than the sieving technique in terms of separating the light fraction for further processing. Of these, pipetting was the most effective technique to reduce the amount of heavy residue accidentally entering the light fraction during separation. Once these samples were separated, both the pipette and decanted samples required further processing to remove the heavy liquid and enable it to be re-used for subsequent samples. This processing added approximately eight hours (dependent on the amount of heavy liquid being recycled) to the laboratory protocol. In contrast, the sieved samples only involved one step of centrifuging and decanting that took around four minutes, before the sample was ready to be reduced and placed on a slide. The process of removing contaminants from the heavy liquid by filtering through a glass mesh, and repeating this step added another five minutes to the protocol before the heavy liquid was ready to be reused. Therefore, despite the initial processing time, the sieving technique is the most time effective and efficient overall method for separating the supernatant and recycling the heavy liquid.

Laboratory processing: Revised starch extraction protocol

After experimentation highlighted potential sources of starch contamination within the materials used during standard starch extraction processes, a method for dispersing charcoal and a new technique for removing the supernatant after density separation and recycling heavy liquid were incorporated into a final extraction protocol. This protocol was based on modification of techniques published by Horrocks (2004), Torrence (2006a, b) and Field (pers. comm. 2012). It involved steps to sub-sample, deflocculate, settling, sieving, centrifuging, density separation, and reduction of samples for slide construction.

Note on sub-sampling and site variation

Talasiu was the first site to be analysed for the presence of starch residues. A 3gm sub-sample was taken from each 5cm spit excavated from the test-pit, and processed to extract preserved starch residues. After radiocarbon dating revealed that all three sites represent relatively discrete time periods, it became clear that there was little intra-site age variation and so few chronological differences would be observed within each of these sites. A decision was made,

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