The experiments that are to be described were as far as possible conducted according to the guidelines stated in section 3.1.5. The organism used was Chlorella vulgaris. The cumulative method given in section 3.1.5.2 was followed; nutrient solution was inoculated with a small amount of an algal culture in the same medium. Under the conditions chosen, this culture became fully developed in 10 - 14 days. At the end of this growth period the entire contents of the dish used for the experiment was evaporated, ashed and returned to the original condition of the initial nutrient solution as described in section 7.1.5.2. For a second growth period this new nutrient solution was inoculated by a culture that had during this time been made ready. This culture was again brought to development, etc... By this means it was possible under the given conditions to repeat the periods of growth six times in, most importantly, the one volume of feeding solution in one and the same vessel.

Since it is important to be able to determine the size of a possible effect after one, two, etc, to six growth periods, the whole experiment was split up into six parallel part experiments, of which the first was broken off after one growth period, the following after two, etc, through to the sixth growth period.

At the same time, six control experiments were set up with entirely the same composition as the experimental group that went with them and for which only the handling after each inoculation differed; they were evaporated and ashed immediately after inoculation. It must be pointed out that this difference in handling does not entirely comply with the demands formulated in section 3.2 for the utmost maintenance of the identity of experiment and control. In later experiments, described below, this difficulty was attempted to be met by the control dishes being at all times during the entire growth period allowed to run together in the apparatus with the actual growth experiments. The algal growth was prevented by means of an artificially brought about acid reaction (experiment III), the exclusion of light (experiment 8508-1), or by heating for one hour at 100 degrees centigrade (experiment V).

[This unnumbered section should perhaps belong elsewhere as it, for example, covers the intended analysis of a number of non volatile chemical elements which, in fact, Holleman did not succeed in doing. It is however, included here because of it's direct relevance to his Chlorella research. See section 6.3, section 7.1.5.4, section 7.2.3 and section 9.1.4.3.]

1. Quantitative composition measured before and after.

2. The experiment to extend as far as possible to the whole life process. Assimilation facilitated by means of (a) air carrying CO2, (b) extra light, (c) agitation.

3. Study only the non volatile elements: K, Ca, Mg, P, (C, H, N, [potentially volatile as elements or compounds]), S(?), Fe(?). Especially study magnesium with regards to the important role of this element in assimilation.

4. [Illegible].

5. Increase of the effect obtained by (a) repetition of culture growth in a solution composed of the same minerals; (b) stimulation of the growth by supply of air - carbon dioxide.

6. Limiting sources of error by; [illegible, incomplete].

Experiments I-III (1975-1982) are given below, as described by Holleman. His later experiments (1982-1989) are described by myself (D.R.C.) based on his laboratory notebooks and other loose notes; these were often very sketchy and difficult to follow and understand.

There were in total 3 series of experiments set up (I-III). The first two consisted of 6 quartz dishes with inoculated nutrient solution and 6 control dishes with similarly handled and inoculated nutrient solution, which were immediately evaporated as described in section 7.1.5.2, ashed and redissolved to produce a solution of the ashes. [See figure 1, section 7.1.1 for an overview of the experimental set-up].

I. The first experiment came no further than the first growth period; a fault in the thermostat of the shaking bath towards the end of this period raised the water temperature to close to 100 degrees. The experimental cultures, in parallel with the controls, were quantitatively worked through to ash solutions. The development of the algae, during nine days of the experiment, was negligible. The likely reason was the small quantity (0.1ml) of inoculation material used. In the following experiments (II and III) the inoculation was thus increased to 2ml of the powerfully developed stock culture. This amount was derived from a paper of van Hille (1930).

II. The second experiment progressed right through to completion, although the development of the algae in the later six stages (a, b, ..., f) was extremely variable.

The ashing caused a considerable alkalisation of the mixture of salts obtained. Therefore, in order to ensure the viability of the algae in the following stages a generous quantity of nitric acid was added to the ash and the excess removed by careful evaporation. For further information see section 7.1.5.2 [Holleman originally referred here to a section which has not been found; it may have shed light on a technical problem encountered during the ashing process that probably affected the results of the experiment enormously - see sections 6.3.2, 10.1.5.2, 10.1.5.4, 10.2.3 for further details]. It appeared during the progress of the experiments that the pH of the newly reconstituted solution depended strongly on the amount of nitric acid added and the method of evaporation. From pre-experiments it was initially concluded that an addition of 0.1m.eq. nitric acid was sufficient. It later turned out that 0.5 to 1.0m.eq. was needed to ensure a pH of 4-6 for the solution attained.

III. The third experiment was in part combined with the second. A place for a culture dish in the shaking bath became free after each stage of experiment II which could be utilised by a new culture. This was made use of in experiment III such that a number of culture and control dishes could go through a number of stages together. The control dish of this experiment was beforehand endowed with the same quantity of nitric acid that the experimental culture received after completion of growth. The idea was to overcome the imperfect parallel treatment of experimental and control cultures and to place both groups open to entirely the same external conditions. Unfortunately it turned out that this quantity of nitric acid (5ml 0.01N) was not sufficient to completely prevent the development of the algae.

A number of intermediary experiments were conducted during this time on the difficulties of analysing for calcium and magnesium. See section 7.2.3 for further details.

Following the unexplained results of experiment II (see section 7.2.1), the experimental procedures were improved and refined. Special attention was given to the prevention of contamination; culture maintenance; ashing and hydrolysis procedures; and the number of experimental replicates (ie parallel running identical cultures). [See figure 1 and figure 7 in sections 7.1.1 and 8.1.1 for details of the experimental set-ups].

Experiment IV cannot be described in any detail because of a severe lack of information recorded in Holleman's laboratory notebook; blank pages were left to be completed at a later date, but this was never done. Whilst the initial set up and most of the results were recorded on loose notes and in his laboratory notebook, neither the aims, nor procedure, nor conclusions were given. It was presumably intended to replicate, or compliment the results of experiment II. Chlorophyll content, phosphate and the elements potassium and sodium (the latter two analyzed by flame photometry) were measured for the 30 (or 24?) cultures. It is not clear whether the number of cycles was 4 or - more logically, given that 6 sets of 4 culture beakers were used - 6. The indications are that they were probably 6 sets of 2 experimental cultures, 1 control and 1 culture for the taking of samples, thus the experiment was presumably run for 6 cycles. Holleman states elsewhere that the results were not positive; ie they followed the conservation laws. The accuracy of the polyphosphate determinations was not thought to be very good.

A large number of relatively small experiments and tests were conducted between experiments IV and V (August '82 -December '85):

- The size and shape of possible culture vessels were examined with the aim, not only of improving agitation, but also with the intention of decreasing the size of the culture vessels to be able to increase the number of vessels that might fit in the shaking bath.

- It was considered that the ashing process, following the Middleton-Stuckey method [presumably the method used so far] may not be sufficient for a complete oxidation of all organic matter. Instead two culture tubes were treated with both nitric and sulphuric acids. In fact [according to Holleman] the sulphate ions would be likely to replace all other anions (except phosphate), which was not acceptable [- would the sulphate affect the algae?]. Therefore further tests on different ashing and hydrolysis procedures (both for the reconstitution of a new culture solution and also for phosphate hydrolysis for analysis) were conducted.

- The consequences of trace element impurities present in the "suprapure" nitric acid were considered to be significant. With each dose of nitric acid, traces of copper would accumulate in the culture solutions. Toxic levels would potentially be reached after only 4 cycles. Whether any practical action was taken was not stated.

- Phosphate, chlorophyll and organic matter determination was investigated to varying degrees.

- The loss of the Labline shaking bath resulted in a major change in apparatus and, to a lesser extent, procedure.

- Agitation of the algae to keep them in suspension was attained by the bubbling of carbon dioxide carrying air directly into the culture fluid. The most suitable culture vessels were test tubes. Temperature was maintained inside a terrarium (an aquarium without water) and was lit by fluorescent tubes designed for use with an aquarium.

- Evaporation and partial ashing of completed algal growth by means of an aluminium block heater for the test tubes was the subject of extensive testing.

- The deficit of potassium measured in experiment II was attempted by Holleman to be brought down to what he called "trivial reasons" [see section 10.2.3 for a critical discussion on this]. According to the literature, polyphosphates, which are formed by heating the ash to 500 degrees, under certain conditions, can form insoluble sodium salts. Holleman put forward the hypothesis that this may also be true for potassium. An analysis of the ash solution by flame photometry requires the absence of any such precipitates. Thus the necessary removal of any precipitates by filtration may result in the measurement of an apparent reduction in the potassium content of a Chlorella culture. The acid treatment of the ash is important in the hydrolysis of the polyphosphates, thus converting them into soluble phosphate ions.

   The experimental procedure was to treat the ashed Chlorella culture with insufficient acid. The unexpected result was that the ash was completely soluble in pure water (at ca.70 degrees). The highly alkaline solution was more so than was expected. The pH was investigated with the titration of hydrochloric acid.

- Another potential cause of the potassium loss recorded in experiment II and considered by Holleman was the involvement of the algal organic matter in the ashing process. The oxidation during the heating to 500 degrees of this organic matter could result in the formation of localised hot spots of a significantly higher temperature. This could lead to the melting, evaporation and loss of some potassium compounds. The [circumstantial] evidence for such an hypothesis was that the potassium loss occurred only with the experimental growth cultures and not in the control dishes in which algal growth did not occur. These former cultures would contain significantly greater amounts of organic matter [this would not, however, explain the potassium's subsequent reappearance].

   The alkalinity of the ash solutions cannot, apparently, be exclusively explained by a removal of water from the phosphates [polyphosphate formation]. Most likely is the removal of nitrates from solution. The nitrates are taken up by the algae out of solution, but there is also the possibility of nitrite formation. Therefore preliminary experiments involving the determination of nitrates were conducted. Unfortunately the results were unreliable due to a suspect spectrophotometer.

- [By way of light relief??] 10 dandelion (Taraxacum) seeds were germinated and subsequently ashed, along with a further 10 control (ungerminated) seeds. Unfortunately there is no record of the ashes ever having been analyzed.

- During (and after) the sprouting of the aforementioned dandelion seeds, Chlorella experiments continued as before. Growth experiments were conducted involving the measurement of algal growth and the monitoring/regulation of the gas supply. The overall gas pressure was able to be better controlled by the addition of a more accurate regulator and a pressure gauge. The supply to each culture tube was able to be controlled by means of adjustable pressure clips attached to the silicon rubber tubing connected to every gas inlet glass capillary tube. The algal growth was monitored every couple of days by measuring the optical density of specially diluted, very small samples of well mixed Chlorella culture solution.

- A proposal to determine the loss of culture fluid due to the possibility of the gas bubbles, bubbling through the cultures, creating spray (micro droplets) was considered, but no clear details or results were given.

- The weight loss of the system was measured so that distilled water could be added to replace that lost by evaporation. This was crucial to the measurements of algal concentration. The necessary uncoupling of the culture tubes from the gas supply was, however, a very delicate operation. Thus it was decided, instead of weighing, to mark the original fluid level on the side of the tube and at the end of the algal growth period, to top up the tube to its original level.

Further modifications to the culture apparatus were made and tested:

- The position of the lights was improved.

- To improve the mixing of the algal suspensions, quartz capillary tubes were compared with very fine PVC and also polythene tubing. The gas pressure, inlet tube internal diameter and also the height of the inlet tube from the bottom of the culture tube all affected the size and rate of bubbles produced. This in turn had an affect on the agitation of the Chlorella which is essential to prevent it's sinking and the formation of clumps.

Foaming of the solutions was another problem. Tests involved:

- Floating a loosely fitting piece of plastic on top of the culture liquid;

- Siliconising the tubes;

- Use of the proprietary solution "Span 85";

- Adding a tiny amount of liquid paraffin to the culture solution;

- Smearing a very small amount of "Vaseline" over the inlet tubes. This latter treatment proved to be the best.

- The evaporative loss of water from the growth cultures was attempted to be compensated for by the warming of the gas wash flask. This successfully increased the water content of the gas supply. In fact when the gas wash flask was heated to 55 degrees the algal cultures actually gained in volume. A "Tecam" thermostatically controlled water bath set to 27 degrees, in which the gas wash flask sat, provided the chosen compromise temperature. The water loss was found to depend on the temperature difference between the laboratory and the cultures.

- The tubing, through which the gas was supplied, was shortened to help reduce water loss by condensation inside the tubes.

Experiment 8508-1 formed a preliminary part of experiment V. Six duplicate culture tubes (ie 12 in total) were inoculated with Chlorella, but with one of these pairs used as a control. The control pair was identical to the others except that light was excluded, thus preventing algal growth. The pairs were stopped after 2, 4, 7, 9, and 11 days. The control pair also underwent the full culture conditions for 11 days. The chlorophyll concentration of each of these tubes was measured by two different techniques. The ash solutions of the two control tubes were later used as part of experiment V.

Before starting experiment V, an experiment was started with the aim of determining that potassium in ionic form is completely detected by flame photometry, independent of the presence of other ions and polyphosphate ions in particular. Holleman here states that these polyphosphates are not only produced during ashing, but also during the physiological process (Kuhl, [?]). The two pairs of culture tubes differed only in the treatment of their ashes. One pair was hydrolysed in the same manner as experiment II with HClO4, the other pair was hydrolysed by the preferred method with HCl. The first pair, treated as in experiment II, possessed a slight precipitate possibly of calcium phosphates. As required for flame photometry the solutions were filtered, thus removing any precipitate from the solutions to be analyzed. The ash solutions were [unfortunately] never analyzed.

Rather than complete his investigations to test for possible trivial reasons for the temporary loss of potassium from the experimental series of experiment II, Holleman set up experiment V, which was designed to be a full repeat of experiment II. All the improvements in apparatus and procedure that had been developed were incorporated (see section 8 for details). The procedure involved maximising the number of replicates and cycles given the constraint that there was room for only 12 tubes in the culture container. The control cultures were killed by a heating to approximately 100 degrees. The growth cultures were then placed alongside the controls in the culture container, both sets of tubes being treated equally. Due to constraints of space the experimental cultures and the controls with which they were paired [ie the experimental and control cultures which were to undergo the same number of experimental cycles] were not able to run at the same time together however. (See section 8.1.5.3 for details).

The observation of strong algal growth in a control tube (originally heated for 2 hours at 100 degrees) led to the immediate setting up of a small experiment. The idea was to test the effectiveness of heat killing the algal cultures. Four culture tubes (using the same stem culture as before) were heated; one for 15 minutes; one for 30 minutes; one tube for 60 minutes and the fourth tube was evaporated till dry then redissolved with distilled water. The first 3 tubes after 14 days showed a certain degree of development. A second experiment was also set up: Holleman had noticed from the literature (Kuhl [?], Walker [?]) that autoclaving, rather than the ultrafiltration used by Holleman, was normally used for the sterilisation of culture media. He considered whether the replacement of this method by ultrafiltration could be the reason for his variable results. Another 4 culture tubes were set up; one pair with autoclaved nutrient solution, the other was not [ultrafiltration was in fact not mentioned here, so I can only assume that it was]. After 4 days hardly any differences were to be observed between them.

Experiment 8611-1 was described as a preliminary experiment to a replication of experiment V. Two parallel experiments with 4 identical tubes in each. The 2 parallel experiments were inoculated each with a different stem culture. The optical density of the cultures was measured at regular intervals. Evaporation of the culture fluid was observed to be considerable. To compensate, a calibrated measuring stick was used to measure the volume of the individual tubes and thus the amount of water lost from each. Approximately 2% of the volume was lost per day. The possibility of the raising of the temperature of the wash flask by a few degrees to compensate for evaporative losses had been tried before and was not reliable. The only practical alternative considered was a return to the weighing, before and after each growth period, of each culture tube and its accompanying quartz glass capillary gas inlet tube.

Four of these tubes were ashed, in 2 stages, in preparation for the next experiment. The tubes were divided into pairs; one set received 0.5ml of 0.1N HNO3 the other set twice as much (2.5x and 5x that used in experiment V, respectively). Both were neutralised using NaOH and topped up with water to 5ml. Their optical density over a period of 12 days was monitored. The first pair showed very good growth, from start to finish. The second pair showed extremely poor growth to begin with, but by the end of the growth period they were very close to having caught up.

Experiment 8712-1 involved the ashing in 2 stages of 5ml each of 5 different stem cultures, of different ages and storage histories, left over from previous cycles of experiment V. These ashes were apparently hydrolysed, as they were recorded amongst the flame photometry potassium and sodium analysis results of experiment V. Nutrient solution, made 3.5 years previously was also analyzed and showed lower potassium values than might be expected. This nutrient solution was presumably that used in experiment V. [Some or all?] of the tubes containing the ashed nutrient solution did however also contain the small rings of silicon rubber tubing used to hold the quartz capillary inlet tubes in place, which had presumably fallen in by accident. Thus the ash was contaminated by silicates. What effect they may have had, if any, was not given. The promised comment on these flame photometry results was never made.

The quartz test tubes showed signs of corrosion from the algal ashing. Also, with the leading of hot air through aqueous solutions which were at boiling point, corrosion was clearly to be seen. The suspension, which presumably consists of pure silicon dioxide, may possibly, by adsorption, have an affect on the chemical composition of the culture solution. The cause of this corrosion may well be (overheated?) steam. By heating too quickly, such steam could be produced by the ashing of the algal carbohydrates and also if such oxidation (burning) occurs before the tubes were completely dry. By means of a phased ashing process, involving the heating being conducted in stages, the water can escape before the burning of the carbon residues. An exploratory experiment was conducted with this in mind: Two quartz test tubes were used, one well used and partly corroded, the other pretty well transparent. Equal amounts of the same algal culture were added, then evaporated till dry (at ca.100 degrees) in the usual manner. On being placed in the oven, detailed observations were made on the ashing process as the temperature increased with time. As usual, the temperature was held for a little over an hour at 500 degrees. The next day again normal procedure was followed for the second ashing at 500 degrees. After half an hour the light colour of the carbon deposits were clearly reduced. After an Easter break [!] the ashing was repeated until all signs of carbon deposits had disappeared. The ash was redissolved and inoculated. Chlorella development was not particularly good. The cultures were then stored in the fridge and the experiment was not taken further. The observations made during the initial heating were potentially significant however. Most of the breakdown (charring) of the organic matter occurred during the heating from 200-300 degrees. Thus a first phase of ashing could take place at 250 degrees for about half an hour. The evaporation procedure was also considered.

A test of the vitality of two stem cultures, one 5 months and the other one year old gave the surprising result that whilst both were still active, the oldest was most so!

Holleman's last laboratory notebook ("Chlorella Research Oct. 1984 - [May 1987] Book V") ended with a consideration of further research involving synchronised Chlorella cultures, though he was somewhat sceptical of the literature on the subject. [The following section shows that this scepticism proved to be unfounded.]

[This last phase of Holleman's work was written on loose notes. My lack of a full understanding of many of the methods and procedures in this section was mostly due to an absence of any explanation by him of these proceedings. In fact, no significant results were recorded during this period which helps explain the enigmatic results of experiment II. Nevertheless, I do not wish to imply that Holleman's synchronisation experiments were not of importance. These last experiments break the mould of much of his previous work. I therefore feel able to take the potentially controversial step of interpreting, rather than directly reporting what I can of that which Holleman wished to be the starting point for further research.]

This section differs from all previous sections in as much as the emphasis is almost entirely on Chlorella itself. Because very little information was recorded on the procedures used, let us start with the principles given in section 3, etc. which have been the basis for all his previous research. Here he states the importance of promoting the conditions necessary for the maximum unfolding of organic life, or development [See section 10.2.2 for a consideration of the complexities involved in such a simple statement]. This was facilitated by examining Chlorella samples from cultures under a microscope. Thus the development of the individual cells was able to be followed. Quantitative cell counts gave an exact measurement of cell density over time. This, coupled with qualitative observations, enabled the progress of external cell development and reproduction to be observed. As usual, the optical densities were also taken; a few times they were compared with total dry weight production. Carbohydrate and protein production were also considered for measurement.

The chemical processes were not entirely neglected; pH as well as nitrate, potassium and sodium were at various times on the programme for analysis.

The reason for Holleman embarking on a journey towards a deeper understanding of Chlorella was certainly his wish to find an explanation for the unexplained appearance and subsequent reappearance of potassium in experiment II. In a letter written in 1991 he stated that for a long time after the results of experiment II [and probably even more so after experiment IV failed to replicate them] he was frightened that he had made a terrible experimental error. It was only later that he developed the idea that a transmutation process may be part of a rhythmic, reversible process [see section 5 and also appendix I]. His earliest recorded ideas on this involved the processes of assimilation and dissimilation [the latter term is not a biological term; at first I considered it to refer the process of respiration which it may well for Holleman have actually become, however in the form in which it was first considered it almost certainly referred to the process of decomposition - see section 10.2.2 for further consideration]. The processes of assimilation and growth occur in the light; respiration and reproduction in the dark. It was the processes of growth and reproduction that Holleman was to focus on by the beginning of this section. The means to this end was the use of synchronous cultures. The method chosen to obtain a homogeneous, synchronised algal culture, with the phases of growth and reproduction in step was fortunately an extremely simple one. It merely involved the implementation of a fixed (regular), light/dark regime of, say, 10 hours light and 14 hours dark.

The literature states that parallel to the light/dark cycle the synchronous cultures should be maintained at a constant cell density by means of a dilution procedure that is in phase with the light/dark regime. This is normally done by counting the number algal cells per millilitre on a daily basis. From this count a dilution of the culture can be calculated to give a fixed cell density. The figure chosen from the literature by Holleman was 1.6 x 10^6 cells/ml. There is no evidence from his notes that such a regular dilution was ever made. Despite the cell count being made fairly regularly, the dilution itself was only recorded as being done at the start of every new culture. In experiment VI however, a dilution may have been conducted at the end of the first growth cycle, immediately before ashing. There is no evidence that these ashes were used though, either to start a new culture or that they were analyzed.

Now it is that I wish to speculate on Holleman's intentions. From the guidelines in section 3, and supported in practice as evidenced by his laboratory notebooks and elsewhere, it is clear that the cumulative method was at the centre of Holleman's transmutation research with Chlorella. Therefore it is probable that his intention was to combine the cumulative method with the production of synchronous cultures. A continuous dilution, in these circumstances, though desirable in maximising the growth of the algae, would involve the continual addition, to the original mineral content, of new nutrient solution; thus any accumulation of the results of a potential transmutation would also be diluted. In cycle one of experiment VI there would have had to have been a dilution of 300 times! (In a later experiment a cell multiplication of over 10,000 times was recorded!!) With the determination of the Chlorella concentration at the end of the first growth period, the required volume of algae required for the inoculation of the next culture cycle may be calculated, removed and temporarily stored. The remaining culture solution may be ashed and redissolved in the usual manner, before being re-inoculated by the Chlorella sample taken earlier. If the timing of the ashing always occurs at the same point in the light/dark regime, any transmutation that may occur during a particular physiological (growth/reproduction, assimilation/respiration) stage of the algae would be accumulated.

The Kalignost quantitative chemical test for the element potassium was re-examined in preparation for ash analyses, but was stopped, presumably because of the moving of his department and his subsequent loss of laboratory space.