2014 Annual Report for FNE14-795
Cultivation of gourmet mushrooms using brewer's spent grain
The Brewery Byproduct Experiment – Mash to Mushrooms
Spent brewers’ grain (SBG) was tested as a substitute for standard spawn substrate and fruiting supplements in the production of Pleurotus ostreatus (Oyster), Lentinula edodes (Shiitake) and Hericium erinaceus (Lion’s Mane) mushrooms at Mycoterra Farm in Westhampton MA. SBG is a waste product of commercial breweries; it is produced in large quantities, and has been previously identified as potential alternative nutrient source in mushroom cultivation. Rye berries are used for the standard spawn substrate at Mycoterra Farm and the standard nitrogen supplement is wheat bran; both are organically certified products sourced through local distributors from Midwest farms. Unable to reliably source SBG from Brewmasters Tavern as planned, spent grain was sourced from Berkshire Brewing Company (BBC) a larger microbrewery located in South Deerfield, MA. As BBC brews batches daily, freshly pressed grain was available for each round. Fresh grain is much better from a contamination-risk perspective as even within hours, potential contaminants can flourish.
The effects of SBG as a substitute for rye grain in the vegetative scale-up and fruiting steps of Oyster, Shiitake, and Lion’s Mane were tested. For Shiitake and Lion’s Mane, the wood lovers, mushroom production involves a vegetative scale-up process where the mycelium progresses from petri dish to first-generation grain spawn (A1) to second-generation grain spawn (A2) to sawdust spawn (A3) to fruiting substrate (Control, B, C1, C2). SBG was substituted for rye berries in the A1 and A2 stages of spawn production for all species. For Oyster mushrooms, the vegetative scale up from A1 to A2 was replicated three times, however each attempt resulted in failure in the treatment batches and the study was unable to observe the final fruiting stages for the Oyster (D). SBG was used as a substitute for wheat bran in the supplemented sawdust mix for Shiitake and Lion’s Mane mushrooms (B, C2).
The roles of our study participants were slightly different than anticipated. Julia Coffey was primarily responsible for inoculations during A1 and A2 stages. Coffey also designed and managed the data collection aspect of the project. The data collection component was a significant time element of the project, requiring ½ to 1 hour daily. Originally it was planned for Dylan Kessler to do these observations, however it proved impractical for him to commute to the site for only a short time daily while Coffey resides on site. Kessler was instrumental in sourcing BSG material and transporting it to the farm weekly. Kessler held a primary role in substrate preparation and sterilization of study materials. Kessler is also completing the data analysis with the technical assistance of Nicholas Brazee of the University of Massachusetts. Chris Haskell was responsible for inoculations during the A3, B and C stages, daily maintenance of the study material in the incubation and fruiting stages and data collection of fresh and dry weights in the B and C study groups.
The objective was to characterize feasibility of substituting brewer’s spent grain (BSG) for rye and wheat bran in the cultivation of three popular edible mushroom species: Oyster (Pleurotus ostreatus), Shiitake (Lentinula edodes) and Lion’s Mane (Hericium erinaceus). The project was divided into four parts, testing the effects of BSG as:
A) A substitute for rye grain in the vegetative scale-up steps, this included three sub-groups: A1- first generation grain spawn; A2 – second generation grain spawn; and A3 -sawdust spawn. Steps A1 and A2 were performed with all species. Lion’s Mane and Shiitake were replicated two times. Oysters were replicated three times. A3 was performed with Shiitake and Lion’s Mane and replicated two times. In A3, control and treatment from A2 was transferred to sawdust bags, which were subsequently used as spawn for the final fruiting stages.
B) Substitution for rye grain in fruiting step of Shiitake and Lion’s Mane. B tested the effects of replacing grain inoculum with BSG on the Biological Efficiency (BE) in the fruiting stages of Shiitake and Lion’s Mane grown on the standard supplemented sawdust mix. The treatment replaced the inoculum with A3 treatment; the control used A3 control sawdust spawn.
C) A replacement for bran as a nitrogen source in the fruiting step of Shiitake and Lion’s Mane. C tested the effect of BSG as a bran replacement on BE in Shiitake and Lion’s Mane. There were two C group treatments: C1 – Control spawn onto SBG supplemented sawdust; and C2 – SBG spawn onto SBG Supplemented sawdust.
The A3 treatment and control were used for multiple replicas in the B, C1 and C2 stages. B and C groups were performed with Shiitake and Lion’s Mane and replicated several times. Fruiting data was collected successfully for four replications for both species, as some attempts resulted in failure in some of the treatment groups.
D) A substitution for rye grain in fruiting step of Oyster mushrooms. Trial D, using Oyster mushrooms, was never achieved due to failure in the A2 stage. For Oyster mushrooms the A1 and A2 stages were completed three times. Due to contamination that pervaded in the A2 stages, the Oyster mushroom trials never made it to the D stage, where SBG was to substitute for the rye inoculum on pasteurized straw substrates. During the subsequent batches of A1 and A2, adjustments were made in attempts to find a viable formula including reducing moisture content and mixing spent grain with rye.
Despite our best efforts, it was determined that spent grain failed as a viable substitute for Oyster mushrooms in our process at Mycoterra Farm. Observation of the Oyster mushroom on the SBG substrate indicated excessive moisture seemed to be the major impediment to the success of this species in colonizing the spent grain.
The experiment started 4/1/14 with petri dish preparation. The timeline outlined in our proposal was adjusted for practical considerations. Instead of running all A1 groups and replicas concurrently, we first started with Lion’s Mane A1; then Oyster as lion’s mane was scaled up to A2; and finally Shiitake as Oyster A1 was moved up to A2. A3 progressed accordingly. Once the first round of A3 spawn had been used for inoculation of the fruiting stage (B/C) or discarded due to incubating too long, we began again at the A1 stage for both Shiitake and Lion’s Mane. This put our schedule back further than anticipated. Final data collection was completed by mid-December.
For each species, the first round of A1 first-generation grain spawn consisted of 18 control jars and 18 treatment jars. The control jars contained 200 g rye grain, 200 g water, and 2 g gypsum. The treatment jars contained identical mass of BSG, 400g, with 2 g gypsum added. Both groups will be inoculated from agar culture.
The second round of A1 spawn consisted of 36 control and 36 treatment jars for Oyster and Lion’s Mane and 30 control and 30 treatment jars for Shiitake. The formulas were repeated as above with greater effort made in reducing moisture content of the SBG. A third round of A1 jars were prepared for Oyster as follows: 17 treatment jars containing: 400g SBG and 2g gypsum; 21 control jars containing: 200g rye grain, 200g water, and 2g gypsum; and a mixed treatment consisting of 16 jars containing 100g rye grain and 300g SBG and 2 g gypsum. Growth was monitored daily and judged on a scale from 0 – 5 until full colonization in each jar was achieved. Frequency of contamination within groups was noted.
A2 involved two treatments and a control. The first treatment (A2.1) involved scaling up first generation grain spawn controls from A1 to spawn bags containing 2400g sterilized SBG with 6g gypsum to create second-generation grain spawn. The second treatment (A2.2) involved scaling up first generation grain spawn derived from the A1 treatment group onto 2400g sterilized SBG with 6g gypsum. The control (A2.3) simply scaled up first generation grain spawn from A1 control group onto 1400g rye prepared with 1000ml water and 6g gypsum.
The first two rounds of Oyster included 9 replicas for A2.1, 11 replicas for A2.2 and 9 replicas of A2.3. The third round of Oyster replicas were increased to 14 replicas for A2.1, 12 replicas for A2.2 and 17 replicas of A2.3. The first A2 round of Lion’s Mane had 8 replicas for A2.1, 11 replicas for A2.2 and 8 replicas of A2.3. The second round of Lion’s Mane had 16 replicas for A2.1, 16 replicas for A2.2 and 16 replicas of A2.3. The first round of Shiitake had 8 replicas for A2.1, 8 replicas for A2.2 and 9 replicas of A2.3. The second round of Shiitake had 15 replicas for A2.1, 15 replicas for A2.2 and 13 replicas of A2.3. Each A2 batch was allowed to incubate 2-4 weeks until full colonization was achieved. Data was measured as in A1.
The control and treatment in A3 each consisted of 40 sawdust bags. The control was inoculated with A2 rye at a rate of 1:6. The treatment replaced rye inoculum with the most successful A2 treatment. Moisture contents ranged 60-70%. Data was measured as in A1, however colonization in individual blocks was not tracked. Each A3 batch was allowed to incubate until fully colonized; it was retained for multiple rounds of inoculation over a several week span. A3 was done in duplicate for both Shiitake and Lion’s Mane. This A3 spawn was used in B and C.
There were some adjustments to the design of the B and C sample groups. In order to be able to compare between the three different treatments they were all prepared simultaneously alongside a control group. Two different supplemented sawdust batches were prepared, one batch our standard supplemented sawdust mix consisting of 8 buckets hardwood sawdust, 2 20kg bags wheat bran and one quart gypsum. This was used for our control group and the B treatment; 20 bags of each were prepared. The other batch included 3 5-gallon buckets of SBG instead of wheat bran. This was used for the C1 and C2 sample groups; 20 bags were prepared from the alternative sawdust mix for each of these groups. Moisture content was adjusted to 60-65% for both the control and treatment groups. All media was autoclave sterilized prior to inoculation. Controls and treatments were incubated in identical environments. Bags were observed daily until full colonization, noting growth rate, colonization time, and contamination rate. Once all treatments achieved full colonization, fruiting was then initiated in the grow room. The first fruiting flush of each batch was harvested over a two-week period and weighed fresh and dried. For both wood-lover species, these replicas yielded four complete data sets of wet and dry yields in the fruiting stages.
Colonization times, contamination rates and yields were all tracked. Colonization progress was tracked using a semi-quantitative 0 (uncolonized)-5 (fully colonized) scale. Completion of colonization on a per bag/jar basis was tracked in A1 and A2. ANOVA was performed for individual colonization times in A1 and A2 in order to determine effect of BSG on colonization times. Fisher’s least significant difference was used to determine significance. Time to complete batch colonization for parts A3, B, C and D was noted, but not tracked on an individual basis and therefore stats will not be performed. Contamination in parts A1-A3 indicated failure of the jar/bag. Contamination in B, C or D was noted, and contamination beyond a low threshold before the first flush completes indicated failure in some treatments. During the colonization period of each step, photographs were used for documentation of notable colonization and contamination data.
The aggregate weight of the first flush was weighed fresh to determine yield of each treatment. Wet weight is effective measure of “financial efficiency” in the marketplace since mushrooms will be sold fresh. The market worth of the mushrooms produced in each treatment will be determined by multiplying batch weight by standard price per pound. In the mushroom industry, Biological Efficiency (BE) is the standard measure of mushroom yield vs. energy input, and is considered essential to evaluating the success of fruiting parameters. The formula is: BE % = (fresh weight of mushrooms / dry weight of substrate) * 100 (Chang et al. 1981). After fresh weights were measured, aggregate dry weights were also measured in order to account for any major differences in water content between treatments.
Analysis of the A1, A2 and A3 stage has been completed. Data has been collected and organized for the B, C1 and C2 fruiting stage, however analysis is still pending on the fresh and dry yields. This data is still being analyzed to determine Biological Efficiency and overall economic viability through a cost-benefit analysis. While we are still in the process of completing our final analysis, we expect this will be complete well before the contract end date. Cost of rye vs. BSG, total colonization times of treatments vs. controls, total yields, contamination rates, and Biological Efficiency are being taken into account. Qualitative measures such as limitations due to BSG spoilage and mushroom quality will also be considered. The ultimate benchmark for feasibility of using BSG in mushroom production is economic viability, however, we will also incorporate the practical considerations of working with SBG observed into our financial assessment.
The study was successful in identifying potential formulas for replacing standard substrates with SBG and ruling out others. Perhaps as significant as the quantitative elements of this study were the qualitative observations made in its course. While the data produced in the study will reveal the economic feasibility, the study experience also exposed other practical considerations in using SBG in mushroom production. In some stages of the vegetative scale-up process, while SBG may be a possible substitute, it is less practical. For example, while we did have success in the substitution of rye berries in both the grain spawn stages (A1&A2), the nature of the spawn made it quite difficult to work with.
The grain was much wetter than anticipated. While it is easy to adjust a dry substrate to a higher moisture content, it is much more difficult reducing moisture content without systems in place to do so. We finally settled on pressing excess moisture out in buckets, however, either a hydraulic press or dehydration system would be necessary to feasibly reduce the moisture content on a production scale. The excess moisture proved most troublesome in the A1 and A2 stages. Obtaining low enough moisture content for a tolerable threshold was particularly challenging for our Oyster strain, Shiitake and Lion’s Mane were both more successful.
In the A1 stage the wetness of the SBG proved physically troublesome. Sticky, wet material made filling the jars difficult. Following inoculation with the agar wedges, it was difficult to distribute the inoculum, and the grain left residue on the jar and filter-patch fitted lids. Such residues are potential sites for contamination to develop, as evidenced in the course of the study: contamination in the SBG jars often began on the grain that adhered to the filter patches. The clumpy nature of the SBG was most cumbersome during the A1 to A2 inoculations. It is critically important these inoculations proceed smoothly and swiftly, with minimal movements and contact. Typically rye grain is loosened by shaking and poured into the open sterilized grain bag with a quick turn of the jar. The clumpy nature of the SBG made swift transfer challenging, often requiring forcible shaking or slamming of the jar over the open bag. Such excess movement can spread contaminants, causing failure in subsequent growth stages. It also added considerable time to the A1 to A2 inoculation process itself.
Impacts and Contributions/Outcomes
Mushroom farming using sterile laboratory technique has an inherent ripple effect whenever a new technique or material is introduced into the production environment. The primary ripples observed were in the form of new contaminants found in both the lab and incubation environment. These included mold, bacteria and yeast organisms. Contaminants were not identified to species but were isolated and removed upon discovery. The primary contaminants usually observed at Mycoterra Farm prior to the project are Trichoderma spp. During the study contamination was first observed in A1 and A2 stages, however as the study drew on, cross contamination occurred with other farm stock. As only the SBG treatments exhibited contaminants at first, it is assumed the origin of the contaminants. Given the microbial nature of brewing itself, it is not surprising that new contaminants correlated with the introduction of SBG into the mushroom farm environment.
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