L.buchneri :The rest of the story

Lawrence R. Jones, PhD

American Farm Products, Inc

ljones@afpltd.net

Lactobacillus buchneri bacteria were originally identified by researchers from buckets of corn silage that stayed surprisingly cool after being exposed to air for several days. These bacteria were isolated and grown in pure cultures to be used as an inoculant. These inoculants are promoted for preventing forage heating and spoilage after exposure to air. Before deciding to use a L. buchneri product, you need to hear the rest of the story.

Chapter 1: Forage stability after being exposed to air is related to forage pH and concentrations of acetic acid levels. In a study to determine what compounds keep forages cool, it was shown that forage stability increases exponentially with acetic acid concentration (Danner, 2003). The higher the acetic acid concentration, the longer the forage stayed cool. What most people do not discuss is how acetic acid actually keep forges stable. The food industry has known for sometime that acetic acid inhibits molds and yeast, which produce heat. However, the second part of the story is that the ability of acetic acid to inhibit molds and yeasts depends on pH. A more acidic environment (i.e., lower pH) results in more acetic acid activity for increasing forage stability (Danner, 2003). For acetic acid, the pH needs to be below 4.79 (i.e., pKa) to get 50% effectiveness for keeping forage cool. For a good description of how acetic acid actually works to inhibit yeasts see Thomas (2002). With this understanding, acetic acid should be corrected for pH level and discussed in terms of “effective” acetic acid. Figure 1 shows examples of forage fermentation expressed as effective acetic acid. Producer take home message: because a low pH (< 4.79) is necessary to make acetic acid highly effective in inhibiting molds and yeasts, a L buchneri bacterial product would not be appropriate for hay crop silage.

Forage pH Acetic acid %DM Effective Acetic acid % DM % Effective
Corn Silage

3.9

3.7

3.25

88%

Corn Silage

4.1

3.7

3.07

83%

Haylage

5.0

3.7

1.41

38%

Haylage

4.5

2.1

1.39

66%

 

Figure 1. Example forages expressed as “Effective Acetic Acid”.

Chapter 2: L. buchneri bacteria must grow to produce acetic acid.L. buchneri bacteria are sensitive to low pH and are slow growing. The industry standard for lactic acid producing inoculants is 100,000 CFU/g of forage. Work at the University of Delaware (Ranjit and Kung, 2000) showed that this level of L. buchneri bacteria was not enough to impact acetic acid or aerobic stability of corn silage. Most commercial products containing L. buchneri bacteria are formulated using at least 400,000 CFU/g in order to out compete native bacteria. This is one reason why L. buchneri based inoculants are typically more expensive per treated ton of forage compared to traditional lactic acid producing inoculants. Researchers (Grazia,1984) have found that L. buchneri bacteria do not grow below a pH of 4.1. In a personal communication, Dr. Muck (USDA-ARS, 2011) indicated that the optimal pH for growth of L. buchneri bacteria is above 5. Consequently, if there is a rapid pH drop, L. buchneri bacteria will be inhibited. In a 2007 presentation, Muck pointed out that the slow growth of L. buchneri bacteria requires 45-60 days of storage to achieve much effect for improving aerobic stability. This slow growth was confirmed by other researchers (Schmidt, 2009). Since acetic acid does not begin to accumulate from the L. buchneri bacteria until 45-60 days, a L. buchneri bacteria product is not appropriate for forages that will be fed shortly (< 60 days) after ensiling.

There are two common products made by L. buchneri bacterial (Oude Elferink, 2001).The first product is acetic acid. Since most untreated forage will normally have 2-2.5% acetic acid, forage treated with L. buchneri bacteria should produce over 2.5% acetic acid to demonstrate effectiveness. The second product is 1,2-propanediol (aka., propylene glycol). This product is only produced by L. buchneri bacteria. Forage samples with an effective L. buchneri fermentation should have 1,2-propanediol concentrations greater than 0.4% DM. See Figure 2 for an example of an effective L. buchneri fermentation. It is important to use a forage testing laboratory that tests for 1,2-propanediol to accurately assess the effectiveness of L. buchneri added to produce acetic acid during fermentation.

Figure 2. Example of corn silage with an effective L. buchneri fermentation. Note the high (> 2.5% DM) acetic acid concentration and presence of 1,2 propanediol. There will be approximately 1% DM loss during this conversion of lactic acid to acetic acid and 1,2 propanediol.

Figure 3 contains an example of corn silage sample with poor L. buchneri fermentation. This is an example of the pH dropping too fast and the L. buchneri bacteria not producing acetic acid. Producer take home message: treating forage with an L. buchneri product should result in > 2.5% DM acetic acid and > 0.4 %DM 1,2 propanediol. But, these products costs energy to produce.

Corn silage with an ineffective L buchneri fermentation. Note the low acetic acid concentration (< 2.5%) which suggests the added L buchneri bacteria did not survive. The low pH and low acetic acid suggest energy and dry matter conservation during fermentation.

Chapter 3: Acetic acid production results in dry matter loss. As discussed above, L. buchneri bacteria convert lactic acid to acetic acid and 1,2 propanediol. In particular, L. buchneri bacteria convert 2 lactic acid molecules into 1 molecule of acetic acid, 1 molecule of 1,2 propanediol and trace amounts of ethanol (Oude Elferink, 2001). All of these end products result in energy loss in the forage. This energy loss is practically important given recent escalating grain prices.

Lactic acid in forage has a ME value of 1.69 Mcal/lb (Knapp, 2012). If you start with 6% DM lactic acid (42 pounds; 70 MCal) before the L. buchneri bacteria begin to convert it to lactic acid, and you end with 3% lactic acid and 2% acetic acid when the L. buchneri bacteria are done, you will need 10 pounds of corn meal per ton of forage to make up the lost energy. The reason for this loss is that L. buchneri bacteria use lactic acid as a food source to make acetic acid. In this process, a 3 carbon molecule (i.e., lactic acid) is converted to a 2 carbon molecule (i.e., acetic acid) and a 1 carbon molecule (i.e., carbon dioxide) is released. It can be shown that there is a 2:1 relationship between acetic acid concentration and fermentation-based dry matter loss. This does not take into account the energy and dry matter loss associated with 1,2 propanediol and ethanol. Producer take home message: there is a 1% DM loss to make 2% acetic acid and 10 pounds of corn meal per ton is needed to replace the energy.

Chapter 4:Formulation of L. buchneri products has a significant impact on their effectiveness. Originally, L. buchneri products contained only these bacteria. Consequently, the early fermentation phase of silage was dominated by native bacteria. Given time (approximately 45 days), L. buchneri bacteria grow and convert accumulated lactic acid to acetic acid. Fermentation losses associated with only L. buchneri bacteria can be enormous. Second generation products include traditional lactic acid producing bacteria, in addition to L. buchneri bacteria, in an effort to control the early phase of fermentation. The problem is that if the early phase is controlled very well (rapid pH drop) then the L. buchneri will not have a good environment in which to grow. It is likely that including a L. plantarum bacteria, which is a traditional silage inoculant, would result in an overly acidic environment unfavorable to L. buchneri bacteria. Furthermore, L. plantarum bacteria are noted for accumulating compounds such as hydrogen peroxide which inhibit most other bacteria. Another approach is to include bacteria from the genus Pediococcus. These bacteria have approximately the same optimal pH range as L. buchneri. Yet, the fact remains that there can be significant dry matter loss in the early fermentation phase if silage pH is not controlled to a low level. Within a week, corn silage should have a pH less than 4 and haylage should have a pH between 4 and 4.7 for maximal dry matter conservation in silage fermentation. Producer take home message: a product that drops pH fast will inhibit L. buchneri bacteria growth.

Chapter 5:The appropriate use of L. buchneri bacteria as an inoculant is when aerobic (exposed to air) heating is not controllable through management. Kung (2010) discussed management approaches to improve aerobic stability of silage. Generally, heating for forages occurs when feed cannot be consumed within 24 hours of air exposure. For example, when a corn silage pile will be move from one location to another. In general, a well packed pile (>50 pounds/ft3) with good face management (> 8” fed/day) will not experience prolonged periods of feed heating. For short episodes, adding propionic acid to the specific heating feed is a possible solution. Commonly, adding 0.1-0.2% (wt/wt) of propionic acid to the feed that is heating (generally corn silage) will inhibit yeast. This equates to 2-4 pounds of propionic acid per ton of forage during the heating episodes. Using a current price of $1/lb for propionic acid, the cost to control heating during feedout would be about $3/ton. This compares to energy costs of $1.50 (10 lbs *$0.15/lb for corn meal) plus the cost of the L. buchneri bacteria product (about ~$1.30/ton). The difference being that the propionic acid is applied only during times of heating while a L. buchneri bacteria product is applied to the entire crop.

Conversely, inappropriate use of L. buchneri bacteria is when native L. buchneri bacteria will naturally produce acetic acid. According to data summarized from Cumberland Valley Analytical Service, corn silage that has a dry matter content of 32% or less will typically have acetic acid content greater than 2.5%. These wet corn silages will naturally have high aerobic stability from this high acetic acid. Adding additional L. buchneri bacteria to wet corn silages is generally not recommended. Producer take home message: only use a L. buchneri product when additional acetic acid is needed to keep forage cool. However, 3 pounds/ton of propionic acid during feedout may be a more cost effective treatment.

Conclusion:L. buchneri bacteria based inoculants generally will not provide the rapid and significant pH drop during the early fermentation phase of a traditional lactic acid producing inoculant. When the pH is left in an elevated range favorable for L. buchneri bacteria, they will grow slowly (> 45 days) to produce acetic acid and 1,2-propanediol. The tricky part of acetic acid is that it will increase the stability of the silage but at a significant fermentation-based dry matter loss. The cost to produce an additional 2% acetic acid is 10 pounds of corn meal. Under current prices, the cost of the L. buchneri bacteria product plus the energy loss will approach $2.80/ton. Due to the added risk of dry matter loss associated with slow or poor fermentation, L. buchneri inoculants should be considered only in scenarios where heating of forages (i.e., aerobic stability) cannot be controlled through management.

An alternative scenario is to focus on minimizing fermentation losses with a lactic acid producing inoculant that rapidly drops pH (~$1.00/ton) to conserve dry matter. During times when this forage has a tendency to heat, add 2-4 pounds of propionic acid per ton of heating forage during feedout to stabilize the forage (~$3/ton). Adding this rate of propionic acid for 220 days has the same cost as the L. buchneri bacteria treatment. Since most farms will not need to add propionic acid for more than 90 days, this options is more cost effective. Once heating become more chronic in nature (> 220 days) the L. buchneri bacteria options is more cost effective.

Finally, references to dry matter loss estimates should be separated into fermentation losses and losses from aerobic stability (i.e., feed out). Some companies will combine fermentation losses with feed out losses for marketing reasons. However, they are controlled separately and should be discussed separately.

References

Danner, H., Holzer, Mayrhuber, E., and Braun, R. 2003. Acetic acid increases stability of silage under aerobic conditions. Applied and Environmental Microbiology 69(1) 562-567.

Grazia, L., Suzzi, G., 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56:373-379.

Knapp, J. 2012. Does Converting Simple Sugars to Lactic Acid Reduce the Energy Available in Silages? Feedstuffs:

Kung Jr, L. 2010. Aerobic stability of silage. Proceeding 2010 California Alfalfa & Forage Symposium and Corn/Cereal Silage Conference. University of California.

Ranjit, N.K. and Kung, Jr, L. 2000. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage. Journal of Dairy Science 83:526-535.

Schmidt, R. J., Hu, W., Mills, J. A., and Kung, Jr, L. 2009. The development of lactic acid bacteria and Lactobacillus buchneri and their effects on the fermentation of alfalfa silage. Journal of Dairy Science 92:5005-5010.

Oude Elferink, S., Krooneman, J., Gottschal, J., Spoelstra, S., Faber, F. and Drlehuls, F. 2001 Anaerobic conversion of lactic acid to acetic acid and 1,2 propanediol by Lactobacillus buchneri. Applied and Environmental Microbiology. 67:125-132

Thomas, K.C., Hynes, S. H., and Ingledew, W.M. 2002. Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids. Applied and Environmental Microbiology 68(4) 1616-1623.