Induced malolactic fermentations and their instigator, ML 34, are celebrating a 50th birthday, and it’s a good time to reminiscence about seminal investigations done on the venerable microbe, Oenococcus oeni, at Hanzell Winery and the University of California, Davis.

While on the topic, it seems fitting to toast the highly skilled winemaker Ralph Bradford (Brad) Webb (1922-99) and his important contributions. By way of giving Webb his instructions after hiring him as winemaker at the fabulously beautiful, brand new Hanzell Winery in Sonoma County, James D. Zellerbach presented a bottle of Romanée Conti Pinot Noir, telling Webb, “This is what I want.”

The winery, nestled among new hillside plantings of Pinot Noir and Chardonnay, and meticulously built in the Burgundy style, rivaled the elegance and promise of Zellerbach’s instructions. Made of native stone, with resident moss kept intact during construction, its walls were maintained lush and verdant with distilled water—tap water would have left an unsightly white crust. The lampshades on interior lights were French double-lobed grape picking baskets.

But much more relevant to his ambitious goals was the custom stainless steel equipment: temperature-controllable fermenters (fabricated to Webb’s specifications) as well as French oak barrels, and nitrogen-protecting bottling capabilities. This impressive combination of quality facilities—along with Webb’s skills and dedication—paid off handsomely. He made superb Pinot Noirs and also, some say, definitive Chardonnays, last year making Wines & Wines’ list of “Wines that Changed the Industry” (January 2009 issue).

Refused to go
But not at first. Webb’s initial Pinot Noir wines, made from purchased grapes, refused to undergo malolactic fermentation, a profound disappointment to him that was as perplexing as it was alarming. Webb knew that the malolactic fermentation (by converting malic acid to less-acidic lactic acid and CO2 as well as adding flavor components) was the sine qua non for conferring the softness and subtle flavors of a quality Pinot Noir that he and Zellerbach sought.

It was perplexing because grapes from the same vineyards regularly underwent the fermentation in other cellars. Even when coaxed by adjusting the pH and SO2 to values generally deemed most favorable, the balky malolactic fermentation still refused to cooperate. It was also unreactive to an increased concentration of lees, achieved by returning to the barrel all lees from the second racking. Similarly, wines from Zinfandel grapes, some of which had undergone malolactic fermentation elsewhere, failed at Hanzell.

Even inoculating wine made at Hanzell with wine that had recently undergone malolactic fermentation at another winery didn’t start ML at Hanzell. Webb was exasperated, but he realized that malolactic-free Hanzell offered a rich opportunity to research the fermentation as well as solve his dilemma.

The malolactic fermentation in California had a long, solid reputation for capriciousness and independence. Most winemakers of the era became aware that the fermentation was under way only when tanks of wine began to rumble softly, usually in mid-winter. Winemakers didn’t start it, and they couldn’t stop it; it just happened. But why only then? The malolactic fermentation seemed to have a mind of it own. It occurred where and when it chose.

Knowing that I (then an assistant professor of enology at UC Davis) was venturing into the microbiology and incidence of the malolactic fermentation in California, Webb dropped by to see me. He offered Hanzell as a negative control for experiments on spontaneous malolactic fermentations, beginning a brief but richly rewarding collaboration as well as a warm friendship.

I had begun my studies by searching for bacteria capable of causing the malolactic fermentation. From samples of dry wines and lees at seven California wineries, I had isolated about 50 strains of lactic acid bacteria (the parent class to which we knew malolactic acid bacteria belong) using Rogosa’s tomato-juice based medium.

The isolates proved to be a diverse lot, representing all four major types of lactic acid bacteria: rods and cocci that did (heterofermentative) and did not (homofermentative) produce gas from glucose. But strikingly, all were able to utilize malic acid in laboratory culture, converting it readily to lactic acid and carbon dioxide. That is, all were metabolically able to mediate a malolactic fermentation, at least in the benign environment of laboratory culture. Malolactic-capable bacteria appeared to be everywhere. But individual samples almost always contained only a single type, suggesting that these bacteria weren’t randomly spread throughout wineries. Only one established itself and became dominant.

Together at Davis, Webb and I evaluated all 50 of these “ML” strains for their ability to grow and remove malic acid from fresh and partially fermented must. Isolates No. 34 and 41, both gas-producing cocci, proved to be effective. We selected number 34, the more vigorous, for further studies, referring to it as ML 34. Although we didn’t publish this information, ML 34 had been isolated from a sample of Barbera (as I recall) that I had taken from a redwood tank at the Louis M. Martini winery in Saint Helena.

Louis P. Martini, son of the founder, was welcoming and fully cooperative, but he preferred that his winery not be identified as the source of this bacterium. Bacteria in wineries didn’t project a positive image. After all, they are the bête-noir, causative agents of Pasteur’s “maladies des vins”. But I’m convinced that today, Martini would not be the least bit reluctant to reveal ML 34’s origin—that he would be proud of his winery’s having been ML 34’s home, where it most probably had resided for years, contributing to the high quality of his red wines. Webb routinely called ML 34 the Martini strain. Alice Webb, Brad’s wife, recently told me that in a drawer in their house she found a test tube with dried contents labeled in Brad’s handwriting, “ML-34, Martini.”

Inoculation succeeds
In 1959, Webb and I took ML 34 to Hanzell to study it under winery conditions. There, we added 100ml of a culture grown in a medium nutritionally enriched with tomato juice to 4 gallons of must that had been fermented to 1° Brix. Two days later, all its malic acid was gone. Then we added 200ml of the lees of this starter culture to a 100-gallon lot of Pinot Noir must that had been fermented to 3° Brix, again initiating a malolactic fermentation that completed 21 days later. We also added 2 gallons of clear wine from the starter culture to 300 gallons of Pinot Noir must also fermented to 3° Brix, and it, too, underwent a malolactic fermentation, also completing in 21 days. At Davis, later during that same vintage, we induced similar malolactic fermentations in partially fermented Cabernet Sauvignon must.

We were delighted. Later in an interview Webb said, “That was the most exciting moment of my life, really.” We were, we believed, the first to induce a malolactic fermentation from a pure culture of a known strain of a bacterium. While preparing our paper for publication, however, we learned that in France, E. Peynaud and S. Domercq had used a similar approach and also been successful, probably a bit before we had. Later we learned from Maynard Amerine, a master of the enological literature, that M. Gomes, J.V.F. da Silva Babo, and A.F. Guimarais had been successful in 1956. But more important than priorities, Webb had taken a long step toward achieving his and his boss’s goal: making the highest quality Pinot Noir wine. And to our satisfaction, we had learned how to control the malolactic fermentation.

What was the key to this control? Why hadn’t the malolactic fermentation occurred spontaneously at Hanzell as it did so readily elsewhere? That seemed obvious enough. There were no resident malolactic bacteria in Hanzell’s new, sanitary facilities. But why had an inoculum from a malolactic-fermented wine failed to induce a fermentation at Hanzell? Certainly, the wine must have contained malolactic bacteria.

We recalled and reasoned from three facts: 1) Spontaneous malolactic fermentations usually started in holding tanks during midwinter, long after completion of the primary yeast fermentation; 2) Species of Leuconostoc, the genus to which we thought ML 34 most comfortably fit, grow only when supplied with a complex set of vitamins and other growth factors. ML 34 most probably needed them, too; 3) Although yeasts, including wine yeasts, don’t require vitamins and growth factors, they are notorious nutritional misers, as I had learned as a graduate student while taking Emil Mrak and Herman Phaff’s yeast course at Berkeley. When yeasts stop growing, they glean growth factors from their surroundings and store them inside their cells in packages called vacuoles, until they nutritionally impoverish their environment.

Added during the primary
Webb and I concluded that it was essential to add (as we did) our inoculum of ML 34 to a must before the primary fermentation was complete, before the yeast were able to strip it of needed growth factors. We also reasoned that was why spontaneous malolactic fermentations started in midwinter. By that time, yeast cells had begun to die, break apart (autolyze) and release these growth factors back into the wine.

One might argue that inoculating before completion of the primary fermentation was effective because the malolactic bacteria required a little sugar to get started. But that wasn’t the answer, at least not the complete one, because we found that adding sugar to a completely fermented wine wasn’t sufficient for it to start the fermentation in these wines. Some proposed that the higher alcohol concentration at the end of the primary fermentation was responsible for preventing proliferation of malolactics. But we had found that successful fermentations followed inoculations at 1° Brix, when the wine’s alcohol content was verging on its maximum.

Others had speculated that initiation of a successful malolactic fermentation depended on the type of yeast used for the primary fermentation. That wasn’t the answer either. Adding our malolactic inoculum to still-fermenting musts produced similarly positive results, regardless of whether the fermentation was being caused by the Montrachet, Burgundy, Tokay, Champagne or Jerez strains of yeast.

Of course, a feasible alternative to inoculating while the primary fermentation is still under way, and a test of our hypothesis, would have been to enrich a fully fermented wine with a complement of growth factors such as those that are now commercially available. We didn’t do that.

No absolutes
Nothing is absolute when dealing with the rich complexity of wines or the numbers and condition of bacterial cells used as inocula for malolactic fermentations. Subsequent investigators have experienced little difficulty starting malolactic fermentations in finished wines. But the ability of yeast to deplete must of essential nutrients for malolactic bacteria has also been established.

ML 34 continued its active participation in winemaking long after I had dropped out and Webb pursued other enological challenges. Gordon Pilone and Ralph Kunkee, who replaced me as microbiologist in the enology department, studied it in greater detail, maintained it in the department’s culture collection, and made it available worldwide. Over the years, ML 34’s name changed, becoming increasingly more elegant and wine-specific. First its species name was changed from the Leuconostoc citrovorum to Leuconostoc oenos. And later it was deemed sufficiently distinct to deserve its own genus, then graduating to is present day status of wine-referenced genus and species, Oenococcus oeni. ML 34 also acquired some competitors, including PSU-1, Enoferm-Alpha, PN4, and MCW.

In spite of their important contributions to wine quality, malolactic bacteria have not escaped suspicion and criticism. As they convert malic acid to softer lactic acid and carbon dioxide, they marginally increase volatile acidity, and they strip the acid groups from some amino acids (decarboxylate them) forming corresponding amines. For example, they convert histidine to histamine and tyrosine to tyramine. These amines induce allergy-like symptoms. Although their concentrations in wine are minute, some speculate that accumulation of histamine and tyramine could present a hazard for persons taking antidepressants that inhibit monoamine oxidase, the enzyme that metabolizes these amines.

On the plus side, we should remind ourselves that the delightful Portuguese vinho verde wines rely on the carbon dioxide from a malolactic fermentation for their slightly sparkling pétillance. It&rs quo;s a mystery why California vintners have never accepted this other gift that malolactic bacteria proffer.

If bacteria capable of the malolactic fermentation can be found almost everywhere—even on our teeth—what’s special about Oenococcus oeni? It’s special because it can grow in wine. It tolerates wine’s low pH and considerable alcohol content. O. oeni probably achieved these abilities rather quickly in evolutionary terms, because we now know that it’s an exceptionally rapid evolver. But that’s another intriguing aspect of its complex and still expanding story. We’re just pleased that it did.

On this 50th anniversary, let’s toast Brad Webb and ML 34 with a glass of California Pinot Noir.

Upon retirement from UC Davis as professor of microbiology, John L. Ingraham took up consultation in biotechnology and writing. His latest book, March of the Microbes, Sighting the Unseen, which takes a birdwatcher’s approach to microbiology, will be released by Harvard University Press in February 2010. He is currently writing a biography of Maynard Amerine. To comment on this article, e-mail edit@winesandvines.com.