SHORT COMMUNICATION
Effect of increasing levels of Chlorella spp. on the in vitro
fermentation and methane production of a corn silage-based diet
Efecto de niveles incrementales de Chlorella spp. sobre la fermentación in vitro y la producción de
metano de una dieta a base de ensilaje de maíz
Efeito do aumento dos níveis de Chlorella spp. na fermentação in vitro e na produção de metano em uma
dieta à base de silagem de milho
Juan de J Vargas ; Federico Tarnonsky ; Araceli Maderal ; Ignacio Fernández-Marenchino ;
Federico Podversich ; Tessa M Schulmeister ; Nicolás DiLorenzo* .
North Florida Research and Education Center. University of Florida. Marianna, FL, USA.
To cite this article:
Vargas JJ, Tarnonsky F, Maderal A, Fernández-Marenchino I, Podversich F, Schulmeister TM, DiLorenzo N. Effect of increasing
levels of Chlorella spp. on the in vitro fermentation and methane production of a corn silage-based diet. Rev Colomb Cienc Pecu
2024; 37(1):42–51. https://doi.org/10.17533/udea.rccp.v37n1a2
Abstract
Background: Generally, the forages used in cow-calf and backgrounding cattle operations have low crude protein and
high fiber concentration, limiting animal performance and increasing greenhouse gas emissions. Chlorella spp., a green micro-
alga, shows promising potential to provide nutrients, especially nitrogen, to low-protein diets. However, information is limited
regarding the effects of Chlorella spp. on the in vitro fermentation and methane (CH4) production of diets. Objective: To evaluate
the effects of increasing inclusion levels of algae (Chlorella spp.) on ruminal in vitro fermentation profile and CH4 production
of a corn silage-based diet. Methods: Incubations were conducted on three separate days using corn silage and gin trash as
substrate (70:30 ratio, respectively). Treatments were control (without algae) and 1, 5, and 10% of algae inclusion in the substrate
replacing the basal diet. Ruminal fluid was collected from two ruminally cannulated Angus crossbred steers fed ad libitum a corn
silage and gin trash diet. Final pH, concentration of volatile fatty acids (VFA) and ammonia nitrogen (NH3-N), in vitro organic
matter digestibility (IVOMD), total gas, and CH4 production were determined after 24 h of incubation. Variables were evaluated
Received: February 3, 2023. Accepted: May 20, 2023
*Corresponding author. North Florida Research and Education Center, University of Florida. 3925 Highway 71, Marianna, FL,
USA, 32446. E-mail: ndilorenzo@ufl.edu
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
eISSN: 2256-2958 Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–51
https://doi.org/10.17533/udea.rccp.v37n1a2
© 2024 Universidad de Antioquia. Publicado por Universidad de Antioquia, Colombia.

43Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–51
https://doi.org/10.17533/udea.rccp.v37n1a2Silage fermentation and Chlorella spp.
using the MIXED procedure of SAS software, and means were compared using orthogonal polynomial contrasts. Results: Algae
inclusion linearly increased (p<0.01) the IVOMD. However, the final pH and concentration of VFA and NH3-N did not differ
(p>0.05) among algae levels. Molar proportion of VFA and the acetate:propionate ratio was not affected (p>0.05) by increasing
algae inclusion. Finally, total gas and CH4 production were not different (p>0.05) among treatments. Conclusion: The inclusion
of Chlorella spp. does not modify the ruminal in vitro fermentation profile nor the CH4 production of a corn silage-based diet.
Keywords: additives; backgrounding systems; cow-calf operations; ensiled forages; green micro-algae; low-protein
diets; methanogenesis; protein supplementation.
Resumen
Antecedentes: Generalmente, los forrajes en los sistemas de producción de cría y levante de ganado tienen baja cantidad de
proteína cruda y alta de fibra, limitando la productividad animal e incrementando la emisión de gases de efecto invernadero.Chlorella
spp., una microalga verde, presenta características promisorias para proveer nutrientes, especialmente nitrógeno, en dietas bajas
en proteína. Sin embargo, existe información limitada relacionada con la inclusión de Chlorella spp. sobre la fermentación y la
producción de metano (CH4) in vitro de la dieta. Objetivo: Evaluar el efecto de incrementar la inclusión de alga (Chlorella spp.) sobre
el perfil de fermentación in vitro y la producción de CH4 de una dieta basada en ensilaje de maíz. Métodos: Las incubaciones fueron
realizadas en tres días diferentes usando ensilaje de maíz y residuo de algodón como sustrato (en relación 70:30, respectivamente).
Los tratamientos fueron: un tratamiento control (sin alga), e inclusiones de 1, 5 y 10% de alga en el sustrato. El fluido ruminal fue
colectado de dos novillos mestizos Angus con cánula ruminal, alimentados con una dieta de ensilaje de maíz y residuo de algodón
a voluntad. El pH final, la concentración de ácidos grasos volátiles (VFA) y nitrógeno amoniacal (NH3-N), la digestibilidad in vitro
de la materia orgánica (IVOMD), y la producción de gas total y CH4 fueron determinadas después de 24 h de fermentación. Las
variables fueron evaluadas usando el procedimiento MIXED del software SAS y las medias fueron comparadas usando contrastes de
polinomios ortogonales. Resultados: Niveles crecientes de alga incrementaron (p<0,01) linealmente la IVOMD. Sin embargo, el pH
final y la concentración de AGV y NH3-N no fueron diferentes (p>0,05) entre los niveles de alga. Además, las proporciones molares
de VFA y la relación acetato:propionato no se afectaron con el incremento (p>0,05) en la concentración de alga. Finalmente, la
producción de gas y de CH4 no fueron diferentes (p>0,05) entre tratamientos. Conclusión: La inclusión de Chlorella spp. no modifica
la fermentación in vitro ni la producción de CH4 en una dieta basada en ensilaje de maíz.
Palabras clave: aditivos; dietas bajas en proteína; forrajes conservados; metanogénesis; microalgas verdes; sistemas de
cría; sistemas de levante; suplementación proteica.
Resumo
Antecedentes: Geralmente, as forrageiras utilizadas em sistemas de criação de bovinos de corte nas fases de cria e recria
apresentam baixa concentração de proteína bruta e alta concentração de fibra, limitando a produtividade animal e aumentando o
impacto ambiental. Chlorella spp. apresenta características promissoras para fornecer nutrientes, especialmente nitrogênio, em dietas
com baixo teor de proteína. No entanto, informações sobre a inclusão de Chlorella spp. na fermentação e produção de metano (CH4)
in vitro ainda são escassas na literatura. Objetivo: Avaliar o efeito do aumento da inclusão de algas (Chlorella spp.) no perfil de
fermentação in vitro e na produção de CH4 em uma dieta à base de silagem de milho. Métodos: As incubações foram realizadas
em três dias diferentes utilizando como substrato silagem de milho e resíduo de algodão (na proporção 70:30, respectivamente). Os
tratamentos utilizados foram: controle (sem alga) e três diferentes níveis de inclusão de 1, 5 e 10% alga no substrato. O fluido ruminal
foi coletado de dois novilhos Angus canulados no rúmen consumindo silagem de milho e resíduos de algodão ad libitum. O pH final, a
concentração de ácidos graxos voláteis (VFA), nitrogênio amoniacal (NH3-N), digestibilidade in vitro da matéria orgânica (IVOMD),
produção total de gás e CH4 foram determinados após 24 h de fermentação. As variáveis foram avaliadas utilizando o PROC MIXED
do software SAS e as médias comparadas por meio de testes polinomiais ortogonais. Resultados: O aumento dos níveis de algas
aumentou linearmente (p<0,01) a IVOMD. No entanto, pH final, concentração de VFA e NH3-N não diferiram (p>0,05) entre os
níveis de algas. Além disso, as proporções molares de AGV e a relação acetato:propionato não foram afetadas pelo aumento (p>0,05)
na concentração de algas. Adicionalmente, a produção total de gás e CH4 também não apresentaram diferenças (p>0,05) em função
dos níveis crescentes de algas. Conclusão: A inclusão de Chlorella spp. não modificou a fermentação in vitro ou a produção de CH4
em dieta à base de silagem de milho.
Palavras-chave: aditivos; conservação de forragem; dietas hipoproteicas; metanogênese; microalgas verdes; operações
de bezerros; sistemas de criação; suplementação proteica.

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https://doi.org/10.17533/udea.rccp.v37n1a2Silage fermentation andChlorella spp.
Introduction
Improving animal performance is a valuable
strategy to promote sustainable agricultural
systems as it provides high-quality protein for a
rapidly growing human population while reducing
the environmental impact per unit of product
(Gerber et al., 2013; Hristov et al., 2013b; Knapp
et al., 2014). In tropical and subtropical regions,
cow-calf and backgrounding operations are
typically maintained with low-quality forages
and limited supplementation, resulting in reduced
animal performance and, subsequently, a more
significant, adverse environmental impact (Pardo
et al., 2009; Beauchemin et al., 2010; Silveira
et al., 2011). Generally, low-quality forages have
reduced crude protein and increased fiber content,
which may impair ruminal fermentation and result
in increased enteric methane emission (Kurihara
et al., 1999; Hess et al., 2003; Tiemann et al.,
2008).
Feed additives have been implemented in
ruminant diets to supply limited nutrients, improve
animal performance, and reduce methane emissions
of nitrogen excretion (Beauchemin et al., 2008;
Leng, 2008; Hristov et al., 2013a). Diets limited
in crude protein require supplemental nitrogen
to promote microbial fermentation, enhancing
energy and metabolizable protein production
(Currier et al., 2004; Leng, 2008). In the current
economy, access to quality protein supplements is
limited due to increased cost and scarce availability
(Tarnonsky et al., 2022); therefore, new, and local
protein sources should be evaluated.
Green micro-algae, such as Chlorella spp.,
exhibit a promising chemical composition for
inclusion in protein-deficient diets. Generally, the
concentration of crude protein and ether extract of
green micro-algae varies between 15 to 60 and 2
to 22%, respectively (Becker, 2007; McCauley et
al., 2020). Early research evaluating the effects of
green micro-algae inclusion in high-quality diets on
modulating lipid biohydrogenation and reducing
CH4 emissions produced contrasting results.
Although green micro-algae supplementation
increased the polyunsaturated lipid content in milk
fat, it reduced dry matter intake affecting animal
productivity and increasing CH4 yield (Moate et
al., 2013). Conversely, green microalgae inclusion
in protein-deficient diets increased nutrient
digestibility, nitrogen flow through the intestine,
and nitrogen retention (Drewery et al., 2014).
Research is limited regarding the supplementation
of green microalgae in silage-based diets on
fermentation parameters and CH4 production;
therefore, this experiment aimed to evaluate the
effects of increasing inclusion levels of Chlorella
spp. on the ruminal in vitro fermentation profile
and CH4 emissions of a corn silage-based diet. It
was hypothesized that increasing the inclusion of
Chlorella spp. in a protein-deficient diet would
improve fermentation and reduce CH4 emissions.
Materials and Methods
Ethical considerations
All animal procedures were approved by the
University of Florida Institutional Animal Care
and Use Committee (#202111460; 14-11-2021).
Location and animal adaptation
This experiment was conducted at the North
Florida Research and Education Center in
Marianna, FL. Two ruminally-cannulated Angus-
crossbred steers (808.8±36.3 Kg of BW) were used
as ruminal fluid donors for the in vitro incubations.
Steers were fed a corn silage, cotton gin trash, and
premix of vitamins and minerals diet (70, 28, and
2% on a dry matter basis, respectively) at least 35
d before collecting ruminal fluid.
Experimental treatment
The diet fed to the steers during the adaptation
period was used as a substrate for the in vitro
incubations. Chlorella spp. algae were provided
dried and pelleted by a local company (Origo,
LLC, Venus, Florida). Diet was dried for 48 h at 55
°C. Corn silage, gin trash, and algae were ground
to pass a 2-mm screen in a Wiley mill (Thomas
Scientific, Swedesboro, NJ) and analyzed for dry
matter (DM), ash, crude protein (CP), neutral
detergent fiber (NDF), and acid detergent fiber
(ADF) at a commercial laboratory (Dairy One
Laboratory, Ithaca, New York; Table 1). In

45Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–51
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addition, algae were analyzed for amino acid and
fatty acid profiles at a commercial laboratory
(Dairyland Laboratories, Arcadia, Wisconsin,
USA) (Tables 2 and 3).
Table 1. Chemical composition (%DM) of ingredients
used in the experiment.
Item Ingredient
Corn silage Gin trash Algae1
Crude protein 8.0 16.0 19.5
Neutral detergent fiber 27.4 61.7 13.6
Acid detergent fiber 16.6 58.8 7.9
Ether extract 3.4 3.2 1.4
Ash 2.8 10.3 54.2
Organic matter2 97.2 89.7 45.8
1Chlorella spp.. 2Organic matter: 100 - % Ash.
Table 2. Amino acid and fatty acid profile of algae
(Chlorella spp.) used in the in vitro incubations.
Amino acid profile Fatty acid profile
(%CP) (g/100 g of fatty acid)
Lysine 5.13 Palmitic acid 38.85
Methionine 1.80 Stearic acid 4.44
Isoleucine 3.60 Oleic acid 30.20
Leucine 8.15 Linoleic acid 16.14
Threonine 5.77 Linolenic acid 10.37
Valine 5.82
Arginine 8.36 Saturated 43.29
Histidine 1.48 Monounsaturated 30.20
Phenylalanine 5.66 Polyunsaturated 26.51
Tryptophan 1.64
Table 3. Calculated chemical composition of the
substrate with increasing algae (Chlorella spp.)
concentration used in the in vitro incubations.
Chemical composition Algae inclusion (%)
0 1 5 10
Crude protein 10.4 10.5 10.8 11.2
Neutral detergent fiber 37.7 37.4 36.1 34.6
Acid detergent fiber 29.3 29.0 27.8 26.3
Non-structural carbohydrates1 43.5 43.3 42.4 41.3
Ether extract 3.3 3.3 3.2 3.2
Ash 5.1 5.5 7.4 9.8
Organic matter2 95.0 94.5 92.6 90.2
1Non-structural carbohydrates: 100 – (% Crude protein + %
Neutral detergent fiber + % Ether extract + % Ash). 2Organic
matter: 100 - % Ash.
Treatments were designed with increasing
proportions of algae in a corn silage and gin trash
mixture (70:30 on a dry matter basis, respectively),
with algae inclusion levels selected to describe a
typical range of additive supplementation in beef
cattle diets. Thus, treatments were as follows:
control without algae and 1, 5, and 10% of algae
inclusion, substituting the corn silage and cotton
gin trash mixture (Table 3).
Rumen fluid collection and in vitro incubations
In vitro incubations were conducted on three
separate days (replicates). Ruminal fluid, collected
from a representative sample of digesta, was
strained through four layers of cheesecloth, placed
in pre-warmed thermos containers, and transported
to the laboratory within 30 min of collection. In
the laboratory, ruminal fluid was maintained under
constant CO2 flux, combined in equal proportions
from the donor steers, and then mixed with
McDougall buffer to a 1:4 ratio of rumen fluid to
buffer (i.e. inoculum).
Treatments were weighed in duplicate into
Ankom bags (0.70 g), heat-sealed, and placed in
a 125-mL serum bottle following the procedure
described by Amaro et al. (2021) with modifi-
cations. Thus, two bottles per treatment were
incubated on each incubation day. Briefly, inoculum
(50 mL) was added to each bottle, including two
bottles without substrate (blanks) under constant
CO2 flux. Bottles were fitted with a butyl stopper,
crimp sealed, and placed in an incubator for 24 h
at 39 °C, set at 60 rpm. At the end of incubation,
before removing the stopper, the final gas pressure
was recorded using a manual transducer (Digital
Test Gauge, Ashcroft Inc., Stratford, CT, USA).
A subsample of gas was collected from the bottle
headspace to determine methane concentration
and was stored in vacuum vials for further CH4
analysis. After removing the stopper, the final pH
of the fermentation fluid was recorded. Two 10-
mL subsamples were collected and acidified by
adding 100 μL of a 20% (vol/vol) H2SO4 solution
and frozen at -20 °C until further analyses. Ankom
bags were removed from the bottles, washed with
tap water until the effluent was clear, dried in a
forced-air oven set at 60 °C for 48 h, and reserved
until further analysis.

Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–5146
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Laboratory analysis
The concentration of VFA in ruminal fluid
samples was determined in a liquid-liquid solvent
extraction using ethyl acetate (Ruiz-Moreno
et al., 2015). Samples were centrifuged for 15
min at 10,000×g. Ruminal fluid supernatant
was mixed with a meta-phosphoric acid (25%
wt/vol):crotonic acid (2 g/L, internal standard)
solution at a 5:1 ratio, and samples were frozen
overnight, thawed, and centrifuged for 10 min at
10,000×g. The supernatant was transferred into
glass tubes (12×75 mm; Fisherbrand; Thermo
Fisher Scientific Inc., Waltham, MA, USA)
and mixed with ethyl acetate in a 2:1 ratio of
ethyl acetate to the supernatant. After shaking
tubes vigorously and allowing the fractions to
separate, the ethyl acetate fraction (top layer) was
transferred to 9 mm-vials (Fisherbrand; Thermo
Fisher Scientific Inc., Waltham, MA, USA).
Samples were analyzed by gas chromatography
(Agilent 7820A GC, Agilent Technologies, Palo
Alto, CA, USA) using a flame ionization detector
and a capillary column (CP-WAX 58 FFAP 25
m×0.53 mm, Varian CP7767, Varian Analytical
Instruments, Walnut Creek, CA, USA). The
column temperature was maintained at 110 °C,
and injector and detector temperatures were 200
and 220 °C, respectively.
The concentration of ruminal ammonia
nitrogen (NH3-N) was analyzed after centrifuging
ruminal fluid samples at 10,000×g for 15 min
at 4 °C (Avanti J-E, Beckman Coulter Inc.,
Palo Alto, CA, USA) following the phenol-
hypochlorite technique described by Broderick
and Kang (1980) with the following modification:
absorbance was read on 200 μL samples at OD620
in flat-bottom 96-well plates (Corning Costar
3361, Thermo Fisher Scientific Inc., Waltham,
MA, USA) using a plate reader (Fisherbrand UV/
VIS AccuSkan GO Spectrophotometer, Thermo
Fisher Scientific Inc., Hampton, NH, USA).
Dried Ankom bags were ashed at 550 °C for 6
h to determine the undigested organic matter on
the remaining fermentation residue. Thus, the in
vitro organic matter digestibility (IVOMD) was
calculated as shown:
IVOMD (%)=[(incubated organic matter
– residual organic matter)/incubated organic
matter]×100
A gas subsample was analyzed to measure CH4
concentration by gas chromatography (Agilent
7820A GC; Agilent Technologies, Palo Alto,
CA, USA). A flame ionization detector was used
with a capillary column (Plot Fused Silica 25m ×
0.32mm, Coating Molsieve 5A, Varian CP7536;
Varian Inc. Lake Forest, CA, USA). Injector,
column, and detector temperatures were 80, 160,
and 200 °C, respectively. Injector pressure was
20 psi with a total flow of 191.58 mL/min and
a split flow of 185.52 mL/min with a 100:1 split
ratio. Column pressure was 20 psi with a flow of
1.8552 mL/min. The detector makeup flow was
21.1 mL/min. The carrier gas was N2, and the run
time was 3 min.
Statistical analysis
Data were analyzed as a randomized complete
block design with three replicates (blocks) using
the MIXED procedure of SAS (SAS Institute
Inc., Cary, NC). Bottles were considered the
experimental unit, and the model included the
fixed effects of algae inclusion and the random
effect of incubation day (replicate). Means were
compared using orthogonal polynomial contrasts.
Significance was declared at p≤0.05.
Results
The inclusion of Chlorella spp. algae did
not affect (p>0.05) final pH, concentration,
or proportion of VFA, acetate to propionate
ratio, the concentration of NH3-N, or
total gas and CH4 production (Table 4).
However, increasing the inclusion of algae
linearly increased (p<0.01) the IVOMD.
Discussion
The chemical composition of algae exhibited
a reduced concentration of NDF and ADF
(13.6 and 7.9%, respectively) and a greater
concentration of ash (51%) compared with a
forage as corn silage (Table 1). A decrease in
dietary fiber content typically results in enhanced

47Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–51
https://doi.org/10.17533/udea.rccp.v37n1a2Silage fermentation and Chlorella spp.
ruminal fermentation. Additionally, previous
studies have reported variable concentration of
ash in Chlorella spp. related to growing condition
and cultivation process modifying the organic
matter concentration (Drewery et al., 2014;
Wild et al., 2019a). In the present experiment,
the decrease in organic matter concentration due
to increasing algae inclusion may have limited
the amount of potentially fermentable material
available (Lodge-Ivey et al., 2014) (Table 3),
resulting in a similar content of non-structural
carbohydrates among treatments (between 40
and 44%) (Table 3), thus potentially explaining
the similar fermentation and lack of differences
in final pH and concentration of VFA.
Additionally, it is expected that comparable
fermentation conditions should not alter the
microbial population or diversity and subsequent
proportion of VFA produced (Murphy et al.,
1982), as was observed in similar studies where
green micro-algae inclusion resulted in little
changes in the concentration of VFA and the
microbial community on in vivo conditions
(Drewery et al., 2014; McCann et al., 2014).
An increase in the concentration of NH3-N
with algae supplementation was observed in an
in vitro fermentation study from Kianai et al.
(2020); however, the concentration of NH3-N was
not affected by increasing algae inclusion in the
present study (Table 4). A similar concentration
of NH3-N among treatments may be explained
by decreased protein hydrolysis or increased
NH3-N utilization by rumen microorganisms
(Sniffen et al., 1992). Algae protein has displayed
greater resistance to hydrolysis by rumen
microorganisms than plant protein (Lodge-Ivey et
al., 2014). The structure and accessibility of algae
protein or the interrelation with other chemical
compounds as phenols may limit proteolysis
during fermentation, resulting in decreased
concentration of NH3-N and possibly increased
the amount of rumen undegradable protein
(Lamminen et al., 2019; Wild et al., 2019b). In
this regard, Drewery et al. (2014) suggested that
algae inclusion may reduce the concentration
of NH3-N in rumen fluid, increase the non-
ammonia nitrogen flow to the intestine, and
reduce the excretion of N in the urine, suggesting
lower protein fermentability in the rumen.
Table 4. Increasing proportion (%) of algae on fluid fermentation pH, the concentrations of volatile fatty acids (VFA)
and ammonia-N, in vitro organic matter digestibility, and gas and methane production in a corn silage-base diet.
Variable1 Algae inclusion (%) SEM P–value2
0 1 5 10 Linear Quadratic Cubic
pH 6.51 6.58 6.58 6.53 0.049 0.926 0.255 0.352
Total fatty acids, mM 28.36 25.89 28.96 26.82 2.844 0.919 0.509 0.201
Acetate, mM 14.75 13.87 15.10 14.25 1.364 0.996 0.524 0.255
Propionate, mM 8.89 7.85 8.78 8.12 0.920 0.774 0.644 0.176
Butyrate, mM 3.51 3.02 3.74 3.27 0.593 0.904 0.385 0.166
Acetate, mol/100 mol 52.01 53.61 52.69 53.18 1.038 0.617 0.885 0.164
Propionate, mol/100 mol 31.06 30.61 30.06 30.42 1.757 0.128 0.066 0.674
Butyrate, mol/100 mol 12.29 11.37 12.64 12.05 1.134 0.698 0.518 0.172
Acetate:Propionate 1.69 1.76 1.78 1.76 0.132 0.396 0.297 0.390
NH3-N, mM 5.34 5.11 4.86 4.34 0.829 0.146 0.983 0.821
IVOMD, % 67.80 67.78 68.85 69.59 0.990 0.002 0.688 0.518
Gas production, mL/g OMd 116.4 100.7 123.9 98.3 13.63 0.339 0.202 0.101
Methane production, mM/g OMd 1.02 0.84 1.05 0.90 0.272 0.827 0.408 0.093
1IVOMD: In vitro organic matter digestibility; OMd: Organic matter degraded. 2Probability of the linear, quadratic, or cubic effect
of increasing the level of algae. SEM: Standard error of the mean; NH3-N: Ammonia nitrogen; IVOMD: In vitro organic matter
digestibility; OMd: Organic matter degraded.

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https://doi.org/10.17533/udea.rccp.v37n1a2Silage fermentation and Chlorella spp.
Conversely, rumen microbes can utilize NH3-N
or preformed amino acids to synthesize microbial
protein (Sniffen et al., 1992). The amino
acid profile of algae demonstrated a greater
concentration of limiting amino acids (Table 2);
however, the amino acid profile shows a lesser
biological value than other feed proteins (Becker,
2007), although it is necessary to determine the
degradability and passage of this algae protein
in further studies. Novel protein sources should
provide an adequate amino acid profile according
to diet characteristics and animal requirements.
Thus, algae could increase the synthesis of rumen
microorganisms providing limiting amino acids
and maintaining a similar NH3-N concentration
after 24 h of fermentation. Nevertheless, this
hypothesis should be tested in future experiments.
In this study, increasing inclusion levels
of algae linearly increased organic matter
digestibility (Table 4). Organic matter digestibility
does not account for mineral concentration in
the substrate and residue; therefore, increasing
organic matter digestibility implies that the
organic fraction of Chlorella spp. has greater
digestibility than the corn-silage and gin trash
mixture. Increasing digestibility should result
in greater VFA concentration, gas production,
or microbial synthesis (Dijkstra et al., 2005). In
this experiment, the algae inclusion did not affect
the concentration and proportion of VFA and
gas production (Table 4). Thus, increasing the
synthesis of microbes could explain the greater
IVOMD maintaining similar VFA concentration
among different levels of algae supplementation.
Algae inclusion did not modify gas and
CH4 production in this experiment (Table 4).
Greater gas production is associated with greater
digestibility when algae from freshwater are
incubated (Dubois et al., 2013). However, reduced
fermentation of the Chlorella spp. relative to
other freshwater algae (Lamminen et al., 2019;
McCauley et al., 2020) and the potential increase
in non-ammonia nitrogen when Chlorella was
supplemented (Drewery et al., 2014) precluded
the possibility of recognizing differences in gas
production. In addition, CH4 is produced according
to the H2 dynamic during fermentation (Janssen,
2010; Ungerfeld, 2015). In this experiment,
the concentration and proportion of VFA did
not differ among algae inclusion; therefore,
it was not expected to observe differences in
CH4 production (Table 4). Production of CH4
is reduced when H2 is redirected to reduced
products (e.g. propionate or microbial synthesis)
instead of methanogenesis (Ungerfeld, 2015) or
due to the presence of secondary compounds
that may affect CH4 synthesis as bromoform or
phlorotannin (Machado et al., 2016; Abbott et
al., 2020). Green microalgae show no secondary
compounds that could affect methanogenesis
(Abbott et al., 2020); however, microalgae have
shown contradictory results on CH4 production
(Fievez et al., 2007; Kiani et al., 2020). The
greater concentration of polyunsaturated fatty
acids (e.g. DHA) explained the CH4 reduction
reported by Fievez et al. (2007) but no by Kiani
et al. (2020). Polyunsaturated fatty acids can
capture H2 during biohydrogenation in the rumen
and show an antimicrobial effect on the rumen
microbial population modifying the fermentation
profile and reducing CH4 production (Johnson
and Johnson, 1995; Beauchemin et al., 2009).
Other algae strains or different cultivation,
harvesting, and processing practices may
increase the antimethanogenic compounds of
green microalgae (Wild et al., 2019a; Kiani et al.,
2020).
In conclusion, including green microalgae
from Chlorella spp. in a corn silage-based diet
does not significantly modify in vitro fermentation
profile and CH4 production. Future research could
evaluate the effect of algae inclusion on rumen
undegradable protein and microbial synthesis
and their technical and economic inclusion
for ruminants in cow-calf and backgrounding
operations.
Declarations
Funding
This study was funded by the North Florida
Research and Education Center of the University
of Florida.

49Rev Colomb Cienc Pecu 2024; 37(1, Jan-Mar):42–51
https://doi.org/10.17533/udea.rccp.v37n1a2Silage fermentation and Chlorella spp.
Conflict of interest
The authors declare they have no conflicts
of interest regarding the work presented in this
report.
Author contributions
JV, ND: conceptualization and experimental
design. JV, FT, AM, IFM, FP: laboratory
procedures and data collection. TMS, ND:
project administration. JV, FT: wrote the
document. All authors have read and agreed to
the published version of the manuscript.
Use of artificial intelligence (AI)
No AI or AI-assisted technologies were used
during the preparation of this work.
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