Blast from the Past – The Known and the Unknown

Santanu

October 06, 2019

It all started a summer afternoon in 1928 when Prof. Alexander Fleming left a petri dish uncapped, forgot to close the window of his laboratory and left town for five weeks and gave mother nature to work out its miracles. On his return, he found that in the petri dish something secreted by a mold (later identified as Penicillin) had lysed and killed the pathogen Staphylococcus. The discovery of Penicillin is a fascinating story as it shows the dual role of serendipity and a prepared mind at it’s full glory. The mold penicillium which secretes the molecule penicillin, grows best at 20 deg C, while for the pathogen Staphylococcus the optimum growth is at 35 deg C. It is speculated that there must have been a snap cold for a week in London during the intense summer of 1928 for the mold to grow and devour the pathogen. And the rest, as they say is history. The discovery of penicillin that ushered in a newer line of treatment in the medicinal use of antibiotics in the 1950’s has undoubtedly bestowed one of the greatest benefits to mankind. Over the following years, the average life span of population increased significantly as starkly exemplified by Ms.Anne Sheafe Miller, who in 1942 was the first patient to be saved by penicillin form a near fatal Streptococcal infection. She lived a full life till her nineties and died of natural cause in 1999.

The Netherlands Yeast and Spirit Factory (NG&SF), located in Delft was one of the oldest fermentation companies in the world. During the Nazi occupation of Netherlands, a period which has been immortalized by the  Anne Frank Diary, unknown, to the world, a team at NG&SF secretly isolated, characterized, and produced Penicillin under the code name “Bacinol”. This was  an  18-month project, that was completely independent of the well known  American and British efforts to produce penicillin. In addition it was achieved under a  war torn chaotic circumstances, with a near zero access to scientific literature,  international scientific discussion and equipment.

Although this story is little known, parts of this narrative are more like components of a thriller than an undertaking in industrial microbiology.

Following the invasion of Netherlands in the summer of 1940, the Queen, the Prime Minister and his cabinet escaped to London.  Hitler then appointed an Austrian civilian administrator over the country to forcibly collect, materials and money, organise captive labour for aiding the Fuhrer’s  war machine. 

The news of Penicillin came to the Dutch probably from the leaflets dropped over Netherlands by the Royal Air-Force of UK.  These were “air magazine” like “The flying Dutchman” newsletters of several pages in miniature format that had information on the  war effort and the progress of the allied forces over the Germans.  The novel discovery of Penicillin convinced F.G.Waller then CEO,  that NG&SF should try to make this fermentation product. This secret project was fortunately  aided by its access to the best fungal culture collection in the world, the Central bureau voor Schimmelculture (CBS) in Baarn

The Dutch microbiologist  evaluated about 23 different fungi, including 18 strains of Penicillium and 3 of Aspergillus. 

To screen for antibacterial activity, scientist  Struyk and Lagendijk seeded plates of peptone agar with a thick suspension of bacteria and added fungal spores. They hoped to find “halos” of bacterial lysis similar to those reported by Alexander  Fleming.  To advance the technique,  the mold was first grown on agar, and then a small plug of agar and mold was placed onto another plate already inoculated with bacteria. Using this modified protocol approach, clear areas were observed around the agar plugs of seven of the tested strains. The one with the greatest antibacterial activity was coded P-6, (Penicillium  baculatum Westling).

It is said.. that the lone German security personnel who was assigned to oversee NG&SF operation was modest in microbiology  and was hoodwinked of the clandestine Penicillin Project.  Moreover he was given a more than generous   helpings with the Dutch Gin Jenever to down his guard. 

In his report, Struyk describes in detail how flasks of P-6 grown for 5 days at 26oC, and the antibacterial agent secreted was soluble in acetone and alcohol. The product was named “Bacinol” as it was produced from Penicillium baculatum. The project name was probably  more borne out of political pragmatism. It was like a smoke screen to keep the Germans unaware of this breakthrough research.

The members of the Delft team then quickly scaled up Bacinol production in an ingenious way due to the lack of instruments and funds. They  decided to use milk bottles as fermentation units which  were available and easy to sterilize. Struyk describes how in a pilot plant room hundreds of milk bottles were stacked for production. To evaluate quality control during production  a quantitative bioassay in which Micrococcus aureus ( the old name of Staphylococcus aureas“strain 6” served as an indicator species and the concentration of Bacinol was expressed in Delftsche Eenheden (D.E.’s or “Delft Units”).

DE was defined as “the amount of bacteriostatic substance which can just completely suppress the growth of the test organism Micrococcusaureus strain 6 in 1 ml of peptone water at 37degC.

After the war in Europe ended, the Delft team  corroborated that “Bacinol” was indeed penicillin. The Delft team scientists concluded that the only detectable difference was that Bacinol was yellowish in color, whereas the American product was white.

The story of Dr. A. Querido, a Jewish physician and NG&SF advisor who along with his family  was incarcerated at Westerbork  concentration camp is an interesting side story of this war time episode. NG&SF managed to declare him as an “essential worker” and, once a month, he was allowed to leave the camp to attend meetings in Delft. While changing trains on one of these trips, he met a colleague from the University of Amsterdam who told Querido that a recent visitor from neutral Portugal had brought a Swiss medical journal with an article by A. Wettstein on penicillin. This information passed on by Dr. Querido  gave further corroboration that the NG&SF team was on the right track. In November 1945 the recovery of Maria Gene, a patient in Delft’s Bethel Hospital signalled the crowning success of the secret production of penicillin.Very little has been written about this extraordinary achievement until the recent research of Marlene Burns.